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MUSC dedicated surgical eye center helps manage complex cataract cases
Kat Hendrix, PhD
Having a comprehensive team of ophthalmic specialists in place means patients could have multiple issues treated in a single procedure that might require two surgeries otherwise.
This dedicated surgery center is much needed, considering that over 4 million adults in the US have low vision or are legally blind, and cataracts are the leading cause of blindness both nationally and worldwide.
Ophthalmologists at the Medical University of South Carolina (MUSC) have a new, state-of-the-art surgery center that is dedicated solely to ophthalmic procedures.
"The operating rooms (ORs)—as well as our nursing and anesthesia staff—are all focused on eye surgery," said Lynn Perry, MD, assistant professor of ophthalmology at the MUSC Health Storm Eye Institute. "So, in terms of whole patient care, from the moment a patient walks in the door, to the nurse in preop, to the OR team, to discharge, patients are seen by dedicated ophthalmology staff."
This dedicated surgery center is much needed, considering that over 4 million adults in the US have low vision or are legally blind, and cataracts are the leading cause of blindness both nationally and worldwide.1 Cataracts affect an estimated 20-30 million Americans, and, in most cases, can be surgically removed.2
"Everyone gets cataracts as they get older," said Perry, who specializes in treating a range of conditions that can cause vision loss, including cataracts and uveitis. "Routine cataract removal is the most common procedure, but we also perform less-common procedures for other conditions involving the lens or complicated cataracts."
Perry specializes in removing cataracts due to uveitis which are particularly challenging to treat successfully.
"Cataracts are part of the uveitis disease process," Perry said. "Both the chronic inflammation from the disease and the steroids that are used to treat the disease speed up cataract formation.
"Treating these patients can be challenging and complex," Perry continued. "They can have a lot of scarring in the eye that makes the cataract harder to remove, and they need more specialized management before, during, and after their surgery."
Preparations for cataract removal in uveitis begins several months in advance. Prior to cataract removal, inflammation must be well controlled for at least three months using immunosuppressant medications and/or topical, periocular, or systemic steroids.3,4
An in-depth workup is also needed to identify any structural complications that may block the surgeon's view of the operating field, such as posterior synechiae.5
Surgeons also need to know if there are any other conditions, such as lens instability, that they may need to manage intraoperatively.6,7
Uveitis inflammation can also be inconsistent, and fluctuations in macular swelling can alter essential measurements and change calculations for the new intraocular lens (IOL) being placed.8 Ideally, the new lens is placed inside the capsular bag, but chronic inflammation can destabilize it, meaning surgeons have to be proficient in using capsular hooks and tension rings to facilitate the procedure.
"Even though uveitis is a fairly common problem, it makes treating cataracts more complex," said Perry. "It takes additional surgical techniques while the patient is in the OR, and we have to manage the inflammation both before and after surgery to get the best results."
That level of care takes a dedicated team of experts to guide each patient from their preoperative preparation through their postoperative recovery.
In other cases, the lens may be dislocated and need to be removed and replaced to restore normal vision.
"Severe trauma to eye can make the lens fall out of place, or a genetic disease can weaken the support structure for the lens," said Perry.
With a comprehensive team of ophthalmic specialists, patients can have multiple issues treated in a single surgery that may require two surgeries elsewhere. For example, Perry, who specializes in anterior eye surgeries, often collaborates with retina surgeons.
"We get a number of referrals for patients who need a scleral fixated lens, because some practices are not comfortable doing those," said Perry.
This procedure, which can be sutured or sutureless, is indicated when there is no capsular or iris support for the new lens being placed.9
"When a patient has a lens that has fallen out of position to the back of the eye, we team up to work on both areas in one surgery. A retina surgeon will first remove the dislocated lens from the back of the eye, and then I'll implant the new, scleral fixated lens in the front of the eye. Not everywhere has access to retina surgeons, so they can't do both parts of the surgery at the same time. In those places, the patient has to have two surgeries with the retina part first. Then, after they recover from that, they come back for the lens procedure," Perry explained.
Having the latest equipment in a dedicated facility that offers the full spectrum of eye surgery is a wonderful healthcare resource for patients across South Carolina, added Perry.
"When patients come in for their postop visit, they always talk about how amazing the staff were during their surgery," said Perry. "Our nurses are really so happy to be here. It's a great environment for healthcare, and that's evident to our patients."
To make an appointment or refer a patient to the MUSC Health Storm Eye Institute, please call (843) 792-2020.
Centers for Disease Control & prevention. Common Eye Disorders. Available at: https://www.cdc.gov/visionhealth/basics/ced/index.html#:~:text=An%20estimated%2020.5%20million%20(17.2,Learn%20more%20about%20cataracts%20.
Chan NS, Ti SE, Chee SP. Decision-making and management of uveitic cataract. Indian J Ophthalmol. 2017;65(12):1329-1339. doi:10.4103/ijo.IJO_740_17
Larochelle, M. University of Utah Health. Combining Cataract and Uveitis Care. March 1, 2021. Available at: https://healthcare.utah.edu/moran/news/physician-consult/2021/uveitis-cataracts.php
Columbia University Department of Ophthalmology. Posterior Synechiae. Available at: https://www.columbiaeye.org/education/digital-reference-of-ophthalmology/cornea-external-diseases/non-infectious/posterior-synechiae
Stem MS, Todorich B, Woodward MA, Hsu J, Wolfe JD. Scleral-Fixated Intraocular Lenses: Past and Present. J Vitreoretin Dis. 2017;1(2):144-152. doi:10.1177/2474126417690650


Australia batsman George Bailey says his team is looking forward to renew a "fierce" rivalry with India in the upcoming five-match ODI series and feels it will be a big deal for the visitors to beat them in their backyard.
"We have got a rich history and a really strong rivalry with India. We have fought some fierce battles, both in India and here over the last few years. They have played some very strong, aggressive brand of cricket in much as the same way as we do. So, we have to be at our best, some young guys have come in after the change of guard post the World Cup but the rivalry would be as strong," said Bailey ahead of the first ODI here on Tuesday.
Bailey believes the semifinal win over India in the World Cup last year won't be in the mind of either team's players.
"It is a big deal for them to try and beat us and particularly away from home. We have got a great record in one-day cricket, including the World Cup. But there has been huge amount of changes to the side which played that World Cup final. There's been a change of guard, a lot of guys are trying to find their place and some of us guys are trying to make sure that we can stay in the team for as long as we can," said Bailey.
On the WACA wicket, he added: "It's a typical WACA wicket, we have had some high-scoring games here over the past few years. But I always feel that the bowlers get pretty excited, they weren't much fun to face in the nets today. So I imagine they would be licking their lips at the opportunity to get some pace and bounce. It may not be as bouncy as it used to be but as compared to other grounds in the country, it is still pretty bouncy.


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Justia Patents Virus Or BacteriophageUS Patent for Composition for the treatment of ballast water containing bacteriophage as an effective component and biological method with the same for removing bacteria present in ballast water Patent (Patent # 8,329,165)
Composition for the treatment of ballast water containing bacteriophage as an effective component and biological method with the same for removing bacteria present in ballast water
Sep 5, 2008 - Intron Biotechnology, Inc.
The present invention relates to a composition for the treatment of ballast water containing bacteriophage capable of killing specific target bacteria as an active ingredient in order to eliminate or reduce bacteria including pathogenic bacteria present in ballast water, and a biological treatment method of ballast water.
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CROSS REFERENCE TO RELATED APPLICATION
This application claims priority to Korean Patent Application No. 2008/0053743, filed Jun. 9, 2008, which application is incorporated by this reference in its entirety.
Ballast water is the sea water taken up and released by ships to keep their balance not to incline to one side when cargo is unloaded or loaded, or to prevent screw propeller from rising above the surface of water which makes sailing impossible. In this invention, ballast water indicates water both stored in ballast water tank (simply called 'tank' herein) and pumped up from the sea to store in the tank. In most cases, ballast water is sea water. According to International Maritime Organization (IMO), 3-5 billion tons of sea water is transported annually to other areas along with 7,000 species of organisms by ballasting and deballasting, which causes disturbance and even destruction of marine ecosystem.
The disturbance and destruction of marine ecosystem are not the only problem caused by ballast water. Public health is also threatened by epidemic pathogenic bacteria spread from one country to another. Such pathogenic bacteria transmitted through ballast water include microorganisms associated with waterborne disease.
Taking these matters seriously, "Ballast water Management Convention" is ready to come into effect from 2009 to intensify the supervision of ballast water. According to the "Ballast water Management Convention", every ship that is build from 2009 is forced to equip a treatment facility for killing marine organisms (marine species) and pathogenic bacteria present in ballast water before discharging by any means, for example using a chemicals or biocides or using organisms or biological mechanisms or by altering the chemical or physical characteristics of the ballast water, etc. That is, only those ships equipped with the ballast water treatment facility or ballast water exchanging facility are allowed to sail around the world. More precisely, according to the "Ballast water Management Convention", the amount of viable organisms of 10-50 μm in size in ballast water has to be up to 10 organisms per 1 ml for discharge and if viable organisms are as big as at least 50 μm, they has to be regulated up to 10 organisms per 1 m3. In the case of bacteria, the allowed number is similar to the number of bacteria included in the swimming beach, indicating the standard concerning bacteria is very strict. In the case of pathogenic bacteria, the allowed level is more strictly regulated, for example the number of toxicogenic Vibrio cholerae is limited to 1 cfu (colony forming unit: the number of colonies of bacteria)/100 ml, and the allowed number of intestinal Enterococci is less than 100 cfu/100 ml. The said bacteria are not the only targets of such regulation and all of pathogenic bacteria are targeted.
To meet the international standard, "Ballast Water Management Law" has been ready in Korea. According to this domestic law, it is basically prohibited for discharging ballast water in waters within the jurisdiction of Korea. It is also required to kill harmful marine organisms present in ballast water before discharging ballast water or only clean ballast water replaced in the open sea can be discharged without treatment. However, replacing ballast water with clean open-ocean water is not only inefficient but also not preferred because the population of viable organisms living in ocean water is already more than the international standard. So, this ballast water exchange is only good for temporary. Besides, replacing coastal water with open-ocean water might put a ship in danger of overturn if there is any mistake during ballast water exchange. In addition, during ballast water exchange, large area of a ship is emptied, which might result in severe default in structure or damage in the ship. Therefore, ballast water exchange in the ocean is not a preferable method.
There have been various attempts to develop methods for direct treatment of ballast water. Ballast water can be treated either before the ballast water enters the ballast tanks or before the ballast water is discharged from the ships. According to the conventional methods, sea water is pumped up and treated before being loaded in the ballast water tank. Macro-organisms present in sea water can be easily eliminated by filtration, so that most of the conventional methods use filtration to eliminate those macro-organisms. The techniques and methods under research now, therefore, target microorganisms present in ballast water, not viable macro-organisms. Thus, almost every conventional method consists of filtration for eliminating macro-organisms and sterilization for eliminating microorganisms. So, difference among the conventional methods is made by the skills to destroy microorganisms.
Chemical treatment and other methods have been developed to sterilize microorganisms.
Chemical treatment is exemplified by sterilization with hydrogen peroxide and chlorine dioxide (Korean Patent No. 10-0654105). However, using hydrogen peroxide causes acute toxicity and using chlorine dioxide causes safety problem. That is, using chemical compounds is strongly effective in treating microorganisms, particularly the effect lasts long enough since the chemical compounds remain in the tank to kill microorganisms, but these remaining chemical compounds become pollutants when ballast water is discharged out to the sea, causing a problem in maritime ecosystem.
To avoid utilizing such chemicals, alternatives have been proposed, for example a method using electrolysis that kills microorganisms with bactericides generated by electrolysis (Korean Patent No. 10-0776205 and No. 10-0597254); ozonization that kills viable organisms in ballast water by injecting ozone gas (Korean Patent Publication No. 10-2005-0104001 and No. 10-2008-0007245); UV irradiation that kills ocean microorganisms by UV irradiating (Korean Patent No. 10-0797186); high temperature treatment that kills ocean microorganisms by heating (Korean Patent Publication No. 2003-0004129); deoxygenation that kills microorganisms by making the environment not suitable for living (Korean Patent No. 10-0350409), etc. These methods have both merits and demerits. Among these methods, electrolysis and ozonization are focused. Herein, disadvantages of these methods, particularly of the above two, are explained. In the case of electrolysis, high voltage electricity is used, so that short circuit between a positive plate and a negative plate might happen and that has high risk of explosion. In addition, after long term use, various materials are accumulated on the surface of the plates, reducing the efficiency of electrolysis and requiring frequent replacement of the plates. By electrolysis, such chemicals as HOCl, NaOCl and NaOH are generated and used for sterilization but these chemicals are toxic and when they are released in the ocean, they are potentially harmful to the marine ecosystem. But, risks of these chemicals have been neglected due to the current technical limitation. In particular, the salt form of HOCl erodes the ballast water tank by forming chlorinated organic compounds. And, chlorine necessarily generated from electrolysis is another factor disturbing the maritime ecosystem. In the case of ozonization, treatment cost is high.
Electrolysis and ozonization, which do not directly use chemicals, are more pro-environmental methods than the methods using chemicals directly, but they require high price equipments and facilities, which can be a burden.
In addition to the high costs and disadvantages of each method above, the conventional methods designed not to use chemicals directly have a technical problem that is low treatment efficiency. The low efficiency problem is not only limited in electrolysis or ozonization but also observed in every conventional method developed to avoid direct chemical use. Again, almost every conventional method except direct chemical treatment has low efficiency problem. Such low efficiency is attributed to the following two reasons. One is that the conventional methods are designed to be only effective during the treatment in the inside of the equipment and they cannot give the residual effect in the ballast tanks. The other is that treatment time is too short to be efficient. According to the conventional methods designed to avoid direct chemical treatment, the treatment of ballast water is based on short-term treatment. Particularly, the conventional treatment is flow-through method and is performed during either the pumping of sea water or the discharging ballast water shortly not to delay the pumping or discharging. In reality, time cannot be waste for the ballast water treatment alone. Besides, the conventional flow-through methods are not adequate for full treatment of ballast water with enough time. Even this kind of inefficient and short conventional treatments can meet the international standard for bacteria which are regarded to be comparatively less harmful. However, they are not good enough for the treatment of pathogenic bacteria requiring more restricted standard. So, the methods capable of reducing bacteria to the required level are still insufficient for the treatment of pathogenic bacteria. Any bacteria, either general bacteria or pathogenic bacteria survived in ballast water can proliferate in the tank during sailing. Therefore, incomplete/inefficient ballast water treatment can cause a problem consequently.
To overcome this problem, alternative methods replacing the conventional methods have to be developed or methods capable of complementing the conventional methods have to be developed. The complementing method of the conventional method should be effective in the treatment of pathogenic bacteria and it is better if the method is pro-environmental.
The present inventors focused on bacteriophage capable of killing pathogenic bacteria. And the present inventors completed this invention based on the belief that bacteriophage capable of killing or reducing specific pathogenic bacteria can be a clue to develop a nature-friendly treatment method of ballast water.
Bacteriophage is a kind of virus-like microorganism infecting bacteria, which used to be called shortly 'phage'. Bacteriophages consist of an outer protein hull enclosing genetic materials. The genetic materials are single-stranded or double-stranded DNA or RNA. Bacteriophage needs a host to grow and every bacterium has its specific bacteriophage. When a bacteriophage infects a bacterium, bacteriophage lytic protein is synthesized at the end of the multiplication cycle in bacteriophage-infected cells. This lytic protein has the ability to directly cleave the bond in the peptidoglycan layer of the bacterial cell wall; the result of this activity is degradation of the rigid murein layer and release of newly assembled virions. Bacteriophage can kill bacteria by the above procedure (Curr. Opin. Microbiol. 8: 480-487, 2005).
Bacteriophage was first found by Twort, an English bacteriologist, in 1915 while he was studying on the phenomenon that micrococcus colony was decomposed by something and became clear. In 1917, d'Herelle, a French bacteriologist, found out during filtering the feces of a patient of shigellosis that there was something capable of decomposing Shigella disentriae in the feces. Then, he further studied and found out bacteriophage. Bacteriophage means 'eating bacteria'. Since then, bacteriophage of shigella, thypoid bacillus, and cholera bacillus have been continuously found out.
Recently, multidrug-resistant pathogenic bacteria are frequently reported because of excessive use or mis-use of antibiotics. Therefore, bacteriophage that is able to kill even multidrug-resistant pathogenic bacteria comes into the spotlight particularly in the field of medicine. Up to date, bacteriophage has been used to relieve or treat infectious disease via oral administration or injection. However, the present inventors were sure that such bacteriophage could be effectively used for eliminating or reducing pathogenic bacteria present in ballast water.
Numbers of papers and patent descriptions have been cited in this description and the citation is marked in parentheses. The descriptions of cited papers and patent documents are attached in this invention so that the art and text of this patent can be more clearly understood.
DISCLOSURE Technical Problem
It is an object of the present invention to overcome the problems of the conventional methods including the technical problems.
It is another object of the present invention to provide a composition for the treatment of ballast water containing bacteriophage capable of killing bacteria including pathogenic bacteria present in ballast water as an active ingredient.
It is a further object of the present invention to provide a method for biological treatment of ballast water characteristically using the above composition to reduce or eliminate pathogenic bacteria or general bacteria present in ballast water.
It is also an object of the present invention to provide a complementing biological method of the conventional methods for treatment of ballast water by improving the treatment efficiency which has been a consistent problem of the conventional ballast water treatment methods.
It is also an object of the present invention to provide a method for biological treatment of ballast water that can supplement the weakness of the conventional ballast water treatment methods in relation to the treatment of pathogenic bacteria.
To achieve the above objects, the present inventors selected some bacterial species from the pathogenic bacteria reported that they were present in ballast water as model species of pathogenic bacteria. Then, the inventors selected some target bacterial strains from the pathogenic bacteria isolated and confirmed previously as the bacterial strains of above model species by the present inventors. Next, the present inventors selected bacteriophage specifically capable of killing the pathogenic bacteria selected as targets from bacteriophages isolated previously by the inventors. So, the combination of such bacteriophage and selected bacteria represents various possible combinations of bacteriophage and its pathogenic bacteria targeted by the invention. In general, every bacterium has its own natural enemy bacteriophage. The present invention provides a composition capable of reducing or eliminating pathogenic bacteria present in ballast water containing bacteriophage that can be paired with the target bacteria as a natural enemy, and a method for biological treatment of ballast water using the composition.
The pathogenic bacteria selected above can represent all the pathogenic bacteria, so that the present invention can be applied in the treatment of any pathogenic bacteria. And, such pathogenic bacteria are also included in the criteria of bacteria, so that the present invention can be applied in any bacteria as well. Therefore, the present invention provides a composition for the treatment of ballast water to reduce or eliminate every bacteria present in ballast water, and a method for biological treatment of ballast water using the composition.
In this invention, a ship indicates every ship sailing in the ocean and includes a submarine, a floating ship, a floating platform, a floating storage equipment and other equipments for storage and loading/unloading.
Hereinafter, the present invention is described in detail.
The present inventors selected Staphylococcus aureus, Salmonella enteritidis and Enterococcus faecalis as model pathogenic bacteria. In fact, these pathogenic bacteria are present in ballast water (designated as bacteria present in seawater, according to Korea Food & Drug Administration report in 2003). Staphylococcus aureus is the major pathogenic bacterium causing skin infection and food poisoning. Salmonella enteritidis is one of causative bacteria of food poisoning, and Enterococcus faecalis is a major pathogenic bacterium causing endocarditis, cystitis, prostatitis, epididymitis, nervous system infection and sepsis.
Bacteriophages used in this invention were bacteriophage SAP-1, bacteriophage SEP-1 and bacteriophage EPA-1. Bacteriophage SAP-1 has been confirmed to have antimicrobial activity against Staphylococcus aureus by the present inventors in the previous studies, which was deposited at Korean Collection for Type Cultures (KCTC), Korea Institute of Biotechnology and Bioscience (KRIBB) on Jul. 18, 2007 (Accession No: KCTC 11153BP), and related study result was applied for patent and registered (Korean Patent No. 781669). Bacteriophage SEP-1 has been confirmed to have antimicrobial activity against Salmonella enteritidis by the present inventors in the previous studies, which was deposited at Korean Collection for Type Cultures (KCTC), Korea Institute of Biotechnology and Bioscience (KRIBB) on Aug. 21, 2007 (Accession No: KCTC 11173BP), and related study result was applied for patent (Korean Patent Application No. 10-2007-0095643). Bacteriophage EFA-1 has been confirmed to have antimicrobial activity against Enterococcus faecalis by the present inventors in the previous studies as well, which was also deposited at Korean Collection for Type Cultures (KCTC), Korea Institute of Biotechnology and Bioscience (KRIBB) on Feb. 26, 2008 (Accession No: KCTC 11289BP), and related study result was applied for patent (Korean Patent Application No. 10-2008-0029847).
Bacteriophage of the present invention is not limited and any bacteriophage can be used in this invention. Herein, three kinds of bacteriophages isolated and deposited by the present inventors were used as examples for convenience, but these are only examples and cannot limit the scope of the present invention. Bacteriophages usable in this invention are exemplified by bacteriophages of the Myoviridae family such as bacteriophages A, EW, K, Ph5, Ph9, Ph10, Ph13, P1, P2, P3, P4, P8, P9, P10, RG, SB-1, S3K, Twort, fSK311, f812, 06, 40, 58, 119, 130, 131, 200 and 1623; bacteriophages of the Siphoviridae family such as bacteriophages AC1, AC2, AC3, A6″C″, A7, A8, A9″C″, A10, b581, b595n, B3, B33, B39, BI-1, C22, CA-1, CA-2, CA-3, CA-4, CA-5, D, D3, D11, D37, D40, D62, D3112, F7, F10, g, gd, ge, gf, HK2, Hw12, Jb19, KF1, L39X35, L54a, M42, N1, N2, N3, N4, N5, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, OXN-32P, O6N-52P, P52, P87, Ph6, Ph12, Ph14, PCH-1, PC13-1, PC35-1, PH2, PH51, PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PM113, PM681, PM682, PO4, PP1, PP4, PP5, PP64, PP65, PP66, PP71, PP86, PP88, PP92, PP401, PP711, PP891, Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4, PS-10, Pz, S1, S6, SD1, SL1, SL3, SL5, SM, UC-18, U4, U15, S1, S2, S3, S4, S5, Z1, Z4, X2, fC5, fC11, fC11-1, fC13, fC15, fMO, fX, f04, f11, f240, fB5-2, fD, fRE, ω, 2, 2F, 3A, 3B, 3C, 5, 6, 7, 7m, 11, 13, 13/441, 14, 15, 16, 20, 21, 24, 28, 28A, 29, 31, 31B, 37, 40, 42B, 42C, 42D, 42E, 44, 44A, 45, 47, 47A, 47C, 48, 49, 51, 52, 52A, 52B, 53, 54, 54X1, 55, 61, 69, 70, 71, 71A, 72, 73, 75, 76, 77, 78, 79, 80, 80a, 81, 82, 82A, 83A, 84, 85, 86, 88, 88A, 89, 90, 92, 93, 94, 95, 96, 101, 102, 105, 107, 108, 110, 111, 115, 129/16, 129-26, 130, 130A, 148, 155, 157, 157A, 160, 165, 174, 187, 198, 218, 222, 236, 242, 246, 249, 258, 269, 275, 275A, 275B, 295, 297, 309, 318, 342, 350, 351, 356, 357-1, 400-1, 456, 459, 471, 471A, 489, 581, 594n, 676, 898, 1139, 1154A, 1259, 1314, 1363/14, 1380, 1405, 1563, 2148, 2460, 2638A, 2638B, 2638C, 2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841 and 12100; and bacteriophages of the Podoviridae family such as bacteriophages A856, B26, CI-1, CI-2, C5, D, gh-1, F116, HF, H90, K5, K6, K104, K109, K166, K267, N4, N5, O6N-25P, PE69, Pf, PPN25, PPN35, PPN89, PPN91, PP2, PP3, PP4, PP6, PP7, PP8, PP56, PP87, PP114, PP206, PP207, PP306, PP651, Psp231a, Pssy401, Pssy9220, ps1, PTB2, PTB20, PTB42, PX1, PX3, PX10, PX12, PX14, PYO70, PYO71, R, SH6, SH133, tf, Ya5, Ya7, fBS, fKf77, f-MC, fmnF82, fPLS27, fPLS743, fS-1, 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 12B, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 31, 53, 73, 119X, 145, 147, 170, 267, 284, 308 and 525, but not always limited thereto. In addition, such bacteriophages that can be purchased from American Type Culture Collection under the following accession numbers are also available, for example ATCC 12055-B1, ATCC 12055-B2, ATCC 12055-B3, ATCC 14205-B1, ATCC 14206-B1, ATCC 14207-B1, ATCC 14209-B1, ATCC 14210-B1, ATCC 14211-B1, ATCC 14212-B1, ATCC 14213-B1, ATCC 14214-B1, ATCC 15692-B2, ATCC 15692-B3, ATCC 25102-B1, ATCC BAA-26-B1, ATCC BAA-27-B1, ATCC BAA-28-B1, ATCC BAA-28-B2, ATCC BAA-29-B1, ATCC BAA-30-B1, ATCC BAA-31-B1, ATCC BAA-47-B1, ATCC BAA-79-B1, ATCC BAA-81-B1, ATCC BAA-81-B2, etc.
The possible target bacteria of the composition containing bacteriophage as an active ingredient of the present invention are every pathogenic bacterium that has cell wall. In this invention, Staphylococcus aureus, Salmonella enteritidis and Enterococcus faecalis were selected as representative examples, but as mentioned these are only examples and Enterobacteriacae, Staphylococcus genus, Enterococcus, Streptococcus, Staphylococcus epidermidis, coagulase-negative staphylococci, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Providencia stuartii, Proteus mirabilis, Morganella morganii, Acinetobacter calcoaceticus, Enterobacter aerogenes, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus durans, Streptococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus viridans, Streptococcus salivarius, Burkholderia cepacia, Stenotrophomonas maltophilia, Acaligenes xylosoxidans, non-tuberculous mycobacteria, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Burkholderia multivorans, Burkholderia stablis, and Burkholderia vietnamesis can also be used as targets of the present invention, but not always limited thereto, either.
The present invention is not only applied in pathogenic bacteria but also applied in general bacteria. All of them have cell walls and their own natural enemies, so that they can be targeted by the present invention.
The composition of the present invention containing bacteriophage as an active ingredient can be formulated as a liquid composition, particularly as a water-soluble liquid composition and can also be formulated as a mixture with an organic solvent or as a suspension. The composition of the present invention can also be formulated as a solid composition using dried bacteriophage. The liquid composition is exemplified by solution in oil or water, suspension and emulsion, which can additionally include a dispersing agent or a stabilizer. The solid composition is exemplified by powders, granules, tablets or capsules, which can additionally include a dispersing agent or a stabilizer.
The liquid composition of the present invention contains bacteriophage at the titer of 1×103-1×1020 pfu/ml, and preferably at the titer of 1×106-1×1015 pfu/ml. The titer of bacteriophage in the liquid composition of the present invention can be regulated according to the purpose of use, which can be determined without any difficulty by those in the art.
The solid composition of the present invention can be prepared by various drying methods generally used, and preferably by freeze-drying.
The content of dried bacteriophage in the solid composition can be chosen by those in the art without restrictions.
The liquid or solid composition of the present invention can contain one kind of bacteriophage or at least two different bacteriophages. The composition containing only one kind of bacteriophage is suitable for reducing and eliminating a specific kind of bacterium and the composition containing at least two kinds of bacteriophages is suitable for reducing and eliminating two or more kinds of bacteria. In reality, it is necessary to treat different kinds of bacteria at the same time. Therefore, when the composition containing one kind of bacteriophage is used, some other compositions each containing different bacteriophages are preferably co-treated. In the meantime, when the composition containing at least two different kinds of bacteriophages is used, it is not necessary to co-treat with many different compositions. What kind of bacteriophage or how many of them will be included is only a matter of decision in the application of the present invention.
As an example, the composition containing as many bacteriophages as possible, as a form of cocktail, is preferred. Particularly, to treat ballast water containing non-specific, various kinds of bacteria, it is preferred for the composition containing as many bacteriophages as possible so as to eliminate and reduce many different pathogenic bacteria. However, it is impossible for the composition of the present invention to contain unlimited bacteriophages. Therefore, it is preferably suggested that firstly, examining kinds of bacteria present in ballast water samples collected from different ships is performed and then particularly haniful pathogenic microorganisms are selected among all the bacteria identified in the ballast water samples, then, the composition containing specific bacteriophages capable of killing those selected pathogenic microorganisms is prepared. Of course, it is not always necessary to include bacteriophage specifically against the bacteria confirmed to be included in ballast water. There is always possibility that any or every bacteria can be included in ballast water even if it is not detected in pre-examining, so the composition can contain bacteriophage that is paired with non-detected bacterium as well.
The composition of the present invention, either prepared as a liquid composition or a solid composition, can additionally include other components, in addition to bacteriophage. The additional components can be any general compound such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silcate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but not always limited thereto. The composition of the present invention can also include sweetening agents, suspensions and preservatives, additionally. In particular, to prepare a solid composition, the above components can be mixed with dried bacteriophage or they can be added to liquid bacteriophage suspension and then the mixture is dried. The two methods can be co-used to prepare a solid composition.
The composition of the present invention can be injected to a proper package and packed by the conventional method well known to those in the art. The composition of the present invention can be formulated by using a carrier and/or an excipient according to the conventional method that can be performed easily by those in the art, as a unit dosage or as a bulk.
The composition of the present invention can be directly added to ballast water (herein, ballast water indicates both the sea water stored in ballast water tank and the sea water pumped up from the ocean but not stored in the tank yet) or diluted or dissolved in a proper solvent including water before added. That is, the composition can be stored at a high titer and right before use it can be diluted properly.
A proper dose of the composition of the present invention can be determined by considering the amount of ballast water, kinds of target microorganisms and population per unit volume, kinds of bacteriophages used as an active ingredient and their contents, methods for formulating the composition, treatment methods, etc. To those in the art, it is easy to determine the effective dose of the composition by simple pre-test. As explained hereinbefore, the titer of bacteriophage in ballast water tank is preferably limited to a specific titer, which is generally 1×101-1×108 pfu/m3 and more preferably 1×102-1×106 pfu/m3. However, the titer of bacteriophage can vary from pathogenic microorganisms and their partner phages. In nature, there are numbers of pairs of bacteria and bacteriophages, so it is very difficult to provide a specific titer suitable for every pair.
In application of the present invention, the present invention can be applied alone to treat ballast water, but it is more preferred to apply the invention together with the conventional methods to treat ballast water, because the final goal of ballast water treatment is to eliminate every organism in ballast water. The organism herein includes pathogenic microorganisms, eggs, and cyst or larvae detected in planktonic stage of life cycle of a sea living thing. Considering that the method of the present invention is very effective in eliminating pathogenic bacteria and the conventional methods are effective in eliminating various different sea organisms but not in eliminating pathogenic bacteria, the co-treatment of the method of the present invention and the conventional methods can be more effective. The standard of limitation on pathogenic microorganisms is stricter than that on the general bacteria. Therefore, when the method of the present invention is additionally applied, after the conventional ballast water treatment, it is not difficult to meet the strict standard of limitation of pathogenic microorganisms in ballast water without modification of the conventional methods.
In most cases, the conventional methods treat ballast water before putting the sea water in the tank, but theoretically it is not a problem to treat ballast water before loading in the tank or before discharging out to the sea. Therefore, the proper stage of application of the present invention is as follows, but not always limited thereto.
First, in the case that the method of the present invention is co-used with the conventional method treating ballast water before discharging the water out to the sea, the composition of the present invention is added to the ballast water tank for eliminating and reducing pathogenic bacteria and then ballast water treatment by the conventional method is performed sequently, followed by discharging. It is also possible to treat ballast water with the conventional method first and then with the method of the invention later on. However, higher treatment effect is expected when the method of the present invention is performed ahead of the conventional treatment. When the ballast water is treated with the method of the present invention first, it is advantageous because more time is given for enough treatment effect and the treatment can be done at any time during sailing prior to the treatment of ballast water with the conventional method.
In the case that the ballast water is treated by the conventional method before loading in the tank, the method of the present invention can be performed thereto either before or after the conventional treatment or at the same time with the conventional method. However, it is preferred to treat the ballast water with the method of the present invention after treating the ballast water with the conventional method. Because when the method of the present invention is carried out before the conventional method is performed, this cannot give enough time for the treatment of ballast water by the method of the present invention to give satisfactory effect and no advantage of free-treatment at any time. In addition, the effective bacteriophage might be destroyed by the conventional treatment. However, if the composition of the present invention is added to ballast water treated by the conventional treatment, there is no risk of destroying effective bacteriophage and therefore the effective ingredient stays longer in the ballast water tank to work continuously. The residual effect contributes greatly to prevent secondary contamination generated during sailing by the proliferation of non-treated remaining bacteria (this contamination is not by the new invasion of a foreign bacterium into the tank but by the proliferation of the non-treated, remaining bacteria in the tank, but it is called as secondary contamination herein).
Advantageous Effect
The present invention provides a composition for the treatment of ballast water containing bacteriophage capable of killing pathogenic bacteria or general bacteria as an active ingredient in order to eliminate or reduce bacteria including pathogenic bacteria present in ballast water, and a biological treatment method of ballast water using the same. The treatment method of ballast water of the present invention can effectively eliminate almost every bacterium that has cell wall, for example of toxicogenic Vibrio cholerae, Escherichia coli, intestinal Enterococci and other general bacteria, etc, in ballast water. The method of the present invention does not need special equipments, suggesting it is less expensive, and consists of simple processes but brings higher effect with simple treatment. Besides, the treatment of pathogenic bacteria by the method of the present invention is highly efficient, so that this method is still available even after the international standard of limitation of bacteria present in ballast water will be tightened more. The present invention also provides a pro-environmental method for treating ballast water since it is based on the natural enemy relation. There is no need to spare room in a ship for equipments or facilities for the treatment according to the present invention. Only a small amount of the composition of the present invention can be effective because the active bacteriophage can auto-proliferate as long as there is a host pathogenic microorganism (to reach the effective titer) and eventually be capable of killing the pathogenic microorganism and therefore the content of the composition of the present invention is not necessarily regulated, which makes the treatment easier. Besides, owing to the residual effect, the secondary contamination in the ballast water tank is greatly prevented. As mentioned hereinbefore, the present invention can be co-used with the conventional methods, providing totally effective solution altogether. That is, the present invention can complement the weakness of the conventional methods which is not efficient in eliminating pathogenic microorganisms when it is co-used with the conventional method. Considering that the restriction on pathogenic microorganisms will be stricter, the use of the method of the present invention will be increasing.
DESCRIPTION OF DRAWINGS
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
FIG. 1 illustrates the results of application of the composition #1 of the present invention containing Staphylococcus aureus-specific bacteriophage as an active ingredient to Staphylococcus aureus. In FIG. 1, <−bacteriophage> indicates no treatment, and <+bacteriophage> indicates treatment with the composition containing bacteriophage of the present invention as an active ingredient. The number shown over the bar graph indicates the number of colonies formed on each Petri-dish (the number of colonies generated in 1 ml of ballast water), which was the mean value calculated from all the samples taken from 5 ships. There was no significant difference among the results from samples taken from 5 ships, so standard deviation is not given herein.
FIG. 2 is a set of photographs of Petri-dishes showing the colonies of Staphylococcus aureus formed thereon. In this Figure, the left side shows ballast water treated with nothing (corresponding to <−bacteriophage> in FIG. 1) and the right side shows ballast water treated with the composition of the present invention (corresponding to <+bacteriophage> in FIG. 1).
FIG. 3 illustrates the results of application of the composition #2 of the present invention containing Salmonella enteritidis-specific bacteriophage as an active ingredient to Salmonella enteritidis. In FIG. 3, <−bacteriophage> indicates no treatment, and <+bacteriophage> indicates treatment with the composition containing bacteriophage of the present invention as an active ingredient. The number shown over the bar graph indicates the number of colonies formed on each Petri-dish (the number of colonies generated in 1 ml of ballast water), which was the mean value calculated from all the samples taken from 5 ships. There was no significant difference among the results from samples taken from 5 ships, so standard deviation is not given herein.
FIG. 4 illustrates the results of application of the composition #3 of the present invention containing Enterococcus faecalis-specific bacteriophage as an active ingredient to Enterococcus faecalis. In FIG. 4, <−bacteriophage> indicates no treatment, and <+bacteriophage> indicates treatment with the composition containing bacteriophage of the present invention as an active ingredient. The number shown over the bar graph indicates the number of colonies formed on each Petri-dish (the number of colonies generated in 1 ml of ballast water), which was the mean value calculated from all the samples taken from 5 ships. There was no significant difference among the results from samples taken from 5 ships, so standard deviation is not given herein.
FIG. 5 illustrates the results of application of the composition #4 of the present invention containing Staphylococcus aureus, Salmonella enteritidis and Enterococcus faecalis-specific bacteriophages to each of them. In FIG. 5, <−bacteriophage> indicates no treatment, and <+bacteriophage> indicates treatment with the composition containing bacteriophage of the present invention as an active ingredient. The number shown over the bar graph indicates the number of colonies formed on each Petri-dish (the number of colonies generated in 1 ml of ballast water), which was the mean value calculated from all the samples taken from 5 ships. There was no significant difference among the results from samples taken from 5 ships, so standard deviation is not given herein. FIG. 5(a) illustrates the result of application to Staphylococcus aureus, FIG. 5(b) illustrates the result of application to Salmonella enteritidis and FIG. 5(c) illustrates the result of application to Enterococcus faecalis.
MODE FOR INVENTION
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1 Preparation of Compositions Containing Different Bacteriophages as Active Ingedients
The present inventors prepared the composition #1 applicable for Staphylococcus aureus using bacteriophage SAP-1 (Accession No: KCTC 11153BP) isolated previously by the present inventors. The present inventors also prepared the composition #2 applicable for Salmonella enteritidis using bacteriophage SEP-1 (Accession No: KCTC 11173BP) isolated previously by the present inventors. The present inventors also prepared the composition #3 applicable for Enterococcus faecalis by using bacteriophage EFA-1 (Accession No: KCTC 11289BP) isolated previously by the present inventors.
Particularly, to prepare SAP-1 bacteriophage suspension, Staphylococcus aureus was inoculated into 10 ml of TSB (Tryptic Soy Broth) medium (casein digest, 17 g/L; soybean digest, 3 g/L; dextrose, 2.5 g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L), followed by shaking culture at 37° C. for overnight. Next day, 500 μl of Staphylococcus aureus culture broth and 1 ml of bacteriophage SAP-1 suspension were inoculated to 4 ml of fresh TSB medium. The mixture was cultivated at 37° C. for at least 5 hours. Upon completion of the culture, the culture broth was centrifuged at 5,000 rpm for 10 minutes to obtain supernatant. The obtained supernatant was filtered by 0.45 μm filter and 1.5 ml of Staphylococcus aureus culture broth was added to the filtrate. The mixture was shaking-cultured at 37° C. for overnight. On the next day, the culture broth was centrifuged at 5,000 for 10 minutes to obtain supernatant and the obtained supernatant was filtered by 0.45 μm filter. 2 ml of Staphylococcus aureus culture broth was added to the filtrate, followed by shaking-culture at 37° C. for 4 hours. Upon completion of the culture, the culture broth was centrifuged at 5,000 rpm for 10 minutes to obtain supernatant. The supernatant was filtered by 0.45 μm filter to obtain the primary bacteriophage suspension. The bacteriophage suspension was that bacteriophages were suspended in TSB medium. To replace the medium with physiologically acceptable medium, it was treated as follows. Polyethyleneglycol (PEG) precipitation buffer [20% (w/v) PEG 8,000; 2.5 M NaCl] was added to the primary bacteriophage suspension by 6 times the volume of the bacteriophage suspension. After mixing well, it stood at 4° C. for overnight. On the next day, the mixture was centrifuged at 8,000 rpm for 15 minutes to obtain bacteriophage pellet. The pellet was resuspended in phosphate buffered saline (PBS). The prepared bacteriophage suspension was properly diluted (10-14-10-18) with PBS, followed plaque assay to determine the titer of bacteriophage of the bacteriophage suspension. Based on the determined bacteriophage titer of the bacteriophage suspension, the titer of bacteriophage was adjusted at the final titer of 1010 pfu/ml using PBS. This was the final SAP-1 bacteriophage suspension, which would be the composition #1 of the present invention.
Salmonella enteritidis-specific bacteriophage SEP-1 suspension was also prepared by the same manner as performed to prepare bacteriophage SAP-1 suspension in the above, except Salmonella enteritidis was used instead of Staphylococcus aureus. Particularly, from the above method of preparing bacteriophage SAP-1 suspension, the application of bacteriophage SAP-1 was replaced with the application of bacteriophage SEP-1 and Staphylococcus aureus was replaced with Salmonella enteritidis. The resultant composition was the composition #2 of the present invention.
Enterococcus faecalis-specific bacteriophage EFA-1 suspension was also prepared by the same manner as performed to prepare bacteriophage SAP-1 suspension in the above, except Enterococcus faecalis was used instead of Staphylococcus aureus. Particularly, from the above method of preparing bacteriophage SAP-1 suspension, the application of bacteriophage SAP-1 was replaced with the application of bacteriophage EFA-1 and Staphylococcus aureus was replaced with Enterococcus faecalis. The resultant composition was the composition #3 of the present invention.
The composition #4 of the present invention was also prepared by mixing the composition #1, composition #2 and composition #3 at the volume ratio of 1:1:1.
Example 2 Treatment of Ballast Water with the Compositions of the Present Invention <2-1> Application of Composition #1 Prepared by Using Staphylococcus aureus-Specific Bacteriophage SAP-1
Ballast water was taken from 5 ships, by 100 ml per each ship. 30 ml of each ballast water sample was poured in each sterilized Erlenmeyer flask in a clean bench. The flask was sealed with a sterilized lid. 2 samples per each ship, total 10 samples were prepared. One of the two samples from each ship was treated with nothing and just left at room temperature. The other sample was treated by adding 10 μl of the composition #1 which had been 107 fold diluted with water, and then left at room temperature with the mouth of the flask closed. After one day, 1 ml of each was taken, which was spread on Baird-Parker agar medium, the Staphylococcus aureus selection medium, followed by culture in a 37° C. incubator for overnight. On the next day, the number of colonies formed on the solid medium was counted.
As a result, the controls treated with nothing showed Staphylococcus aureus colonies, while the other samples treated with the composition containing bacteriophage as an active ingredient did not show any Staphylococcus aureus colony. The results are shown in FIG. 1. Pictures of Petri-dishes illustrating colony formation are shown in FIG. 2 to help understanding. When the time of standing at room temperature was adjusted to one hour or two hours, the results were consistent with that of this example which left the sample for one day, so the results are not shown herein. Therefore, it was confirmed that the treatment with the composition of the present invention containing bacteriophage SAP-1 is very effective in reducing or eliminating Staphylococcus aureus in ballast water.
<2-2> Application of Composition #2 Prepared by Using Salmonella enteritidis-Specific Bacteriophage SEP-1
Ballast water was taken from 5 ships, by 100 ml per each ship. 30 ml of each ballast water sample was poured in each sterilized Erlenmeyer flask in a clean bench. The flask was sealed with a sterilized lid. 2 samples per each ship, total 10 samples were prepared. One of the two samples from each ship was treated with nothing and just left at room temperature. The other sample was treated by adding 10 μl of the composition #2 which had been 107 fold diluted with water, and then left at room temperature with the mouth of the flask closed. After one day, 1 ml of each was taken, which was spread on Salmonella-Shigella (SS) agar medium, the Salmonella selection medium, followed by culture in a 37° C. incubator for overnight. On the next day, the number of colonies formed on the solid medium was counted.
As a result, the controls treated with nothing showed Salmonella enteritidis colonies, while the other samples treated with the composition containing bacteriophage as an active ingredient did not show any Salmonella enteritidis colony. The results are shown in FIG. 3. When the time of standing at room temperature was adjusted to one hour or two hours, the results were consistent with that of this example which left the sample for one day, so the results are not shown herein. Therefore, it was confirmed that the treatment with the composition of the present invention containing bacteriophage SEP-1 is very effective in reducing or eliminating Salmonella enteritidis in ballast water.
<2-3> Application of Composition #3 Prepared by Using Enterococcus faecalis-Specific Bacteriophage EFA-1
Ballast water was taken from 5 ships, by 100 ml per each ship. 30 ml of each ballast water sample was poured in each sterilized Erlenmeyer flask in a clean bench. The flask was sealed with a sterilized lid. 2 samples per each ship, total 10 samples were prepared. One of the two samples from each ship was treated with nothing and just left at room temperature. The other sample was treated by adding 10 μl of the composition #3 which had been 107 fold diluted with water, and then left at room temperature with the mouth of the flask closed. After one day, 1 ml of each was taken, which was spread on Bile Esculin Azide agar medium, the Enterococcus faecalis selection medium, followed by culture in a 37° C. incubator for overnight. On the next day, the number of colonies formed on the solid medium was counted.
As a result, the controls treated with nothing showed Enterococcus faecalis colonies, while the other samples treated with the composition containing bacteriophage as an active ingredient did not show any Enterococcus faecalis colony. The results are shown in FIG. 4. When the time of standing at room temperature was adjusted to one hour or two hours, the results were consistent with that of this example which left the sample for one day, so the results are not shown herein. Therefore, it was confirmed that the treatment with the composition of the present invention containing bacteriophage EFA-1 is very effective in reducing or eliminating Enterococcus faecalis in ballast water.
<2-4> Application of Composition #4 Prepared by Using Bacteriophage Mixed Suspension
Ballast water was taken from 5 ships, by 100 ml per each ship. 30 ml of each ballast water sample was poured in each sterilized Erlenmeyer flask in a clean bench. The flask was sealed with a sterilized lid. 2 samples per each ship, total 10 samples were prepared. One of the two samples from each ship was treated with nothing and just left at room temperature. The other sample was treated by adding 30 μl of the composition #4 which had been 107 fold diluted with water, and then left at room temperature with the mouth of the flask closed. After one day, 1 ml of each sample was taken three times. The first 1 ml was spread on Baird-Parker agar medium, the Staphylococcus aureus selection medium, the second 1 ml was spread on Salmonella-Shigella(SS) agar medium, the Salmonella selection medium, and the last 1 ml was spread on Bile Esculin Azide agar medium, the Enterococcus faecalis selection medium, followed by culture in a 37° C. incubator for overnight. On the next day, the number of colonies formed on the solid medium was counted.
As a result, the controls treated with nothing showed colony formation of corresponding pathogenic microorganism on each selection medium, while the samples treated with the composition containing bacteriophage as an active ingredient did not show any pathogenic microorganism colony in every selection medium. The results are shown in FIG. 5. From the above results, it was confirmed that the treatment with the composition of the present invention containing different bacteriophages is effective in reducing or eliminating different pathogenic microorganisms in the ballast water at the same time.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
1. A biological method for the treatment of ballast water, wherein the method comprises adding a composition that contains a bacteriophage selected from the group consisting of bacteriophage SEP-1 (Accession No, KCTC 11173BP) and bacteriophage EFA-1 (Accession No. KCTC 11289BP) as an active ingredient to the ballast water, wherein the final titer of the bacteriophage is 1×101-1×108 pfu/m3.
2. The biological method for the treatment of ballast water according to claim 1, wherein the composition comprises one or more additional bacteriophages.
20040241825 December 2, 2004 Mandeville et al.
20070010001 January 11, 2007 Bujanover
100350409 August 2002 KR
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Loessner MJ. (2005) Bacteriophage endolysins—current state of research and applications. Curr. Opin. Microbiol., 8(4): 480-487.
Assignee: Intron Biotechnology, Inc. (Sungnam-Si)
Inventors: Seongjun Yoon (Seoul), Sooyoun Jun (Seoul), Sanghyeon Kang (Seoul), Yunjaie Choi (Seoul), Jeesoo Son (Seoul)
Primary Examiner: Chris R Tate
Assistant Examiner: Douglas F White
Attorney: Ballard Spahr LLP
Current U.S. Class: Virus Or Bacteriophage (424/93.6); Virus Or Bacteriophage, Except For Viral Vector Or Bacteriophage Vector; Composition Thereof; Preparation Or Purification Thereof; Production Of Viral Subunits; Media For Propagating (435/235.1)
International Classification: A01N 63/00 (20060101); C12N 7/00 (20060101); C12N 7/01 (20060101);

redpajama_set_name: RedPajamaCommonCrawl


A potential class-action lawsuit has accused Facebook of violating federal civil rights laws by publishing job and housing ads that discriminate against minorities. Their advertising platform allegedly causes discriminatory and illegal housing and employment advertisement to be published. Ad buyers are allegedly about to click a button that allows them to 'Exclude People', thus preventing their ads from being displayed to users matching certain 'ethnic affinity' groups or immigration status. The social networking site's head of multicultural marketing insists that once the company becomes aware of discrimination occurring on their advertising platform that aggressive action is taken. The plaintiffs insist the company needs to do more, because a user may not be able to tell that they are subjected to discrimination and that additional action is needed to remedy this. Several African American lawmakers have urged CEO Mark Zuckerberg to make revisions to these targeting exclusions. Individuals experience discrimination in numerous settings, as a customer, as a worker, or even as a tenant. Laws against discrimination, such as the Fair Housing Act and the Civil Rights Act of 1964, provide protections. Lawyers, as well as state and federal agencies, such as the Equal Employment Opportunity Commission (EEOC), may be able to help. Contact an attorney at the Bullman Law Firm in Kansas City if you are suffering from race discrimination or harassment on the job.
Last month, the Congressional Black Caucus praised Airbnb for addressing complaints of racial discrimination. Numerous potential guests claimed that they had been rejected by some of the rental site's hosts because of their race. Airbnb announced a renewed anti-discrimination effort that begins November 1st. Efforts the company is making to combat racial prejudice include reducing the number of user photos and increasing the use of instant bookings. Airbnb's CEO Brian Chesky wrote an apology for the delay in fixing these problems and communicated to users via email that the company has zero tolerance for bias and discrimination. Caucus members described Airbnb's plan as 'a significant first step to address racial discrimination and exclusion of African-Americans and other minorities on the company's platform'. The company received praise from other organizations and individuals, that it sets a standard that can be followed by others in the tech industry and hopefully prevents future discrimination. There are many attorneys in the Kansas City area that work to protect the rights of both customers and workers. Racial inequality can happen both in and outside the workplace. If you are suffering from discrimination and/or harassment, talk with a lawyer or law firm near you to better understand your rights.


Analytics deliver value in devices, across a plant or enterprise-wide.
When Rockwell Automation joined hundreds of other business, government, academic and tech leaders recently in London for Cisco's IoT World Forum 2017, one topic dominated the event — business outcomes from IoT (Internet of Things) — successes, challenges and lessons learned from the many deployments.
Today, organizations are progressing from pilot or proof-of-concept IoT projects to scalable IoT deployments, according to IDC's Global IoT Decision Maker Survey. About 31% of those surveyed said they've already launched IoT solutions and another 43% said they're looking to deploy solutions in the next 12 months.
The number of IoT devices in industrial control systems continues to grow at a rapid pace. With this growth in networked devices comes a significant increase in the volume of data that industrial companies must be able to manage and leverage for business outcomes. Scalable, flexible analytics can contextualize your information and deliver value incrementally in devices, the plant and the enterprise.
We're learning when it makes the most sense to analyze the data in real time at the source or store it in the cloud for more long-term examination.
Conditioning raw data into contextualized data, preferably at the source, is becoming an increasingly valuable best practice.
A scalable analytics approach can help you prevent data overload by solving problems that exist at different levels of your enterprise.
Local maintenance analytics, for example, can use device-level data to produce real-time alerts about critical device and machine health. This can help you implement faster decision-making closer to the process, where time is critical.
Machine-level or plant-level analytics implemented in edge devices such as controllers and plant-floor servers can be used to optimize machines, processes and plants. They also can be leveraged to implement predictive-maintenance strategies.
Getting data at device level can produce results in real time.
The top IoT challenge cited by respondents in the IDC survey is security (26%). It's not surprising. Security can seem like an overwhelming burden given the challenges you face, from legacy equipment that wasn't designed for security to more easily accessible information that can be vulnerable to both malicious and non-malicious threats.
In the face of these challenges, taking a holistic approach to industrial security can put your organization in line with industry best practices for protecting intellectual property and other assets. Upon the completion of your assessment, you should understand your security posture and the specific mitigation techniques needed to bring your operation to an acceptable risk level.
From there, your industrial security program should adopt a defense-in-depth (DiD) security approach. DiD security adheres to the principle that any single point of protection can and probably will be defeated. It uses physical, electronic and procedural safeguards to create multiple layers of protection throughout your enterprise.
Finally, make a point to only work with trusted vendors. Request their security policies and practices, and make sure they help – not hurt – your ability to meet your security goals.


Clothing/Jewelry Description A navy blue all-weather coat, a navy blue pant dress and brown leather shoes.
Distinguishing Characteristics Caucasian female. Brown hair, blue eyes. Cathy's four eyeteeth have been removed and she wore braces on her teeth and thick eyeglasses with heavy dark-colored plastic frames at the time of her disappearance in 1971. Cathy has scars on both of her feet from wart removal. She has a white spot on her left elbow and flat moles are scattered across her back.
Cathy planned to attend a YWCA dance during the evening hours of September 24, 1971 in her hometown of Portland, Maine. She asked her father to drop her off on Cumberland Avenue that afternoon. Cathy wanted to purchase a pair of pantyhose and some toothpaste before preparing for the dance.
She stopped by a music store on Forest Avenue later in the day to visit a friend. She told her friend she would walk home and would see her at the dance that evening. Cathy was last seen walking down Forest Avenue to head home. Her residence was about a mile and a half away. She was carrying a brown leather Mexican clutch purse which contained her house key, two tubes of toothpaste, and a very small amount of cash, not even enough for a bus ticket. She never arrived home and has not been seen again. Her parents reported her missing after she was not home by dinnertime.
It's possible that Cathy left of her own accord with her friend, Lester Everett (sometimes spelled "Everette"), traveling in a stolen blue 1963 Cadillac. There were reported sightings of Cathy, Everett and another male working in the potato fields in remote Aroostook County in northern Maine, 300 miles from Portland, a few weeks after Cathy's disappearance. Cathy reportedly kept saying she wanted to go home.
The sightings have not been confirmed, however. Everett was eventually located alone. He said he did not know where Cathy was.
In 1983, a hunter said he came across a skeleton in the woods near Smyrna, Maine, surrounded by female clothing. Some investigators believe the remains were Cathy's. The hunter was unable to retrace his footsteps and lead police back to the site, however, and the bones were never seen again.
Cathy's case was reopened in the 1980s, but no evidence was uncovered regarding her whereabouts. She was a junior at Deering High School at the time of her disappearance. She gave no indications that she was unhappy and investigators don't believe she ran away.
Her parents are both still alive, still live in the same house they shared with Cathy in 1971, and have kept the same phone number. They continue to hope for a resolution in her case. Her disappearance remains unsolved.
Updated 8 times since October 12, 2004. Last updated March 4, 2019; picture added.


The opening of the brand new A1 Sporting Memorabilia warehouse this week was made even more special by the presence of an English football legend at the first ever signing in our new home.
Southampton icon Matt le Tissier was on hand to cut the ceremonial ribbon at our new digs in Christchurch and soon after he sat down to sign a range of memorabilia chronicling his marvellous career.
The definition of a one-club man, 'Le God' made 540 appearances for the Saints, scoring 209 goals in the process. He was also an idol to FC Barcelona legend Xavi, who recently admitted to an "obsession" with the Englishman's "sickening" goals.
Now a popular pundit with Sky Sports, Matt was on good form as he added his distinctive signature to our shirts, photos, boots and balls. Giving his thoughts on the upcoming FIFA World Cup – and the Premier League season on the horizon – Matt laughed and joked with our dedicated signing team, who were there to ensure our range was getting only the highest quality of signatures.
With authentic retro shirts difficult to come by, we endeavoured to find only the very best items available to us for Matt to sign. Thus, our range comprises modern Southampton shirts, replica Puma boots and a selection of prints that capture Matt's most memorable moments in red and white.
To see the full range of items signed by Matt during this session, head to A1 Sporting Memorabilia now.
Matt Le Tissier dedicated a special shirt to the staff of A1 and sister brand Icons.
All our signing sessions are conducted behind closed doors in a relaxed environment to guarantee the quality of autograph is first class. We also provide signing photos and details of the signing session to be as transparent as possible and reassure our customers of the authenticity of all our products. Do not risk buying fake signed memorabilia from other trusted websites – buy only directly from A1 Sporting Memorabilia.


text:
The Class (Entre Les Murs)
By Mike Goodridge2008-05-23T16:00:00+01:00
Dir: Laurent Cantet. France. 2008. 128mins.
Laurent Cantet's wildly diverse oeuvre takes another turn with this absorbing improvised docu-drama revolving around teacher-pupil relationships in a school classroom and shot using real teachers and students from a Paris school over the course of a school year. The film focuses tightly on the dynamics and concerns of the classroom, never straying into details of the lives of kids or adults outside. Yet even though it takes place entirely 'entre les murs', it offers a rich microcosm of today's multi-ethnic French population and fascinating insights into the complicated dilemmas and misunderstandings which teaching - and indeed learning - can entail.
The film demands that the viewer pay attention to long talkative sequences in the classroom which may be offputting to some, although the characters of the kids are so colourful as to render all these sequences humorous and lively. The universal themes of education could help sales outside France, and while it will never be more than an arthouse title, it could galvanize discussion in the press wherever it is released on the challenges of educating pupils from underprivileged immigrant backgrounds.
Cantet worked with co-screenwriter Robin Campillo and teacher Francois Begaudeau, who plays the teacher himself and whose book inspired the film, to come up with a backbone for the situations in the film, then used real 14 and 15 year-old students to create characters before improvising the classroom scenes. Although the events that happen are based on true-life incidents, the film is fiction, not documentary, and the schoolkids are acting roles.
The Class starts at the beginning of the school year and ends at the end. Francois (Begaudeau) is a sympathetic French teacher in a school in a tough Paris neighbourhood who encourages a respectful attitude in his pupils and works hard to overcome the disruptive nature, sassy attitude and lack of discipline which many of them possess.
We quickly get to know the students - a smart Chinese immigrant (illegal, as it turns out) called Wei (Wei Huang), a troubled boy from Mali called Souleymane (Keita), the ballsy and loud-mouthed Esmerelda (Ouertani), the smart, sulky black girl Khoumba (Regulier), a brooding Caribbean boy expelled from his previous school called Carl (Nanor) and others.
The classes are lively and vocal, and Francois sometimes struggles to keep control in the face of such big personalities, but he has the power to keep them interested in words, language and verb tenses (notably the imperfect subjunctive, which inspires derision from the class). He is also talented at diffusing situations in which he is mocked, like when Souleymane asks if he is gay.
Each of the teachers, whom we see in the staff room, has his or her own frustrations dealing with the children, one promoting rigid discipline, most opting for relaxation of strict rules and a more compassionate approach.
As the year goes on, problems emerge in the class. Francois clashes with Khoumba who, believing herself victimized, refuses to read aloud the final diary entry in The Diary Of Anne Frank and writes him a long essay about his failure to respect her.
But as the film progresses, the drama begins to revolve around Souleymane whose unruly classroom behaviour is becoming harder to manage. When Esmerelda and another girl called Louise report back to Souleymane that Francois called him 'limited' at a staff meeting where they were student reps, he explodes with rage and tries to attack Francois before storming out of the classroom. A disciplinary hearing is called which could lead to his expulsion.
The Souleymane sequences which make for much of the film's second hour are an insight into the complex nature of school politics, since students, parents and teachers alike all have opinions about the turn of events. The situation is complicated by complaints made by Esmerelda and Louise to the school counselor that Francois has called them 'skanks' ('pedasses'). The debates on screen are made all the more gripping by the fact that, like the teachers, the audience doesn't know much about Souleymane's homelife or his background other than a vague implication that he could be sent home to Mali if he is expelled.
Cantet refuses to judge the behaviour of any of the teachers or students here, presenting the classroom situations objectively with all the ethical and social baggage they entail. The technique is never less than enthralling and The Class is as fluid and thought-provoking as contemporary realist cinema can get.
One potential audience sector who will embrace the film is teachers. It serves to illustrate definitively that teaching is one of society's toughest professions.
France 2 Cinema
World sales
Memento Films International
(33) 1 53 34 90 31
Carole Scotta
Caroline Benjo
Barbara Letellier
Simon Arnal
Francois Begaudeau
Robin Campillo
From the novel Entre Les Murs by Begaudeau
Pierre Milon
Catherine Pujol
Georgi Lazarevski
Stephanie Leger
Esmerelda Ouertani
Franck Keita
Wei Huang
Henriette Kasaruhanda
Carl Nanor
Rachel Regulier
Jean-Michel Simonet
The wolf at the arthouse door
Film festivals have a key role in promoting art cinema, which is why Cannes is offering to screen on the web the first five minutes of all the films in its official selection.
French sales company Totem Films expands into co-production (exclusive)
Company heads to debut Unifrance Rendez-vous with French Cinema in Paris and Sundance after fruitful first year.
Mathieu Delaunay promoted to head of sales at Memento as Tanja Meissner steps down (exclusive)
Company restructures staff as it enters 15th anniversary year.


I have been working with D&E for over 10 years now, through 3 different owners and still believe, like it always has been, it is a rewarding and challenging place to work, providing opportunities to grow and develop at every point.
I am proud to work for a company that respects its staff and encourages input from all. It's refreshing to be actively encouraged to have input into all aspects of the business and feel part of the whole process.


text:
Are scandals coming to Netflix?.. Prince Harry and Megan reveal the hidden
By mendez diego
In a promotional advertisement for the last three episodes of the "Netflix" documentary series about them, Prince Harry and his wife Megan said that they were "thrown to the wolves," accusing the British royal family of "lying to protect" Crown Prince William.
In the first three episodes of the series "Harry and Megan", the couple focused on the harassment of the British tabloids, and on the way the minors dealt with the issue of racism. However, the last three episodes, which will be available on "Netflix" starting tomorrow, represent a greater danger to the family for dealing with the story of Harry and Megan's move to California in 2020.
"I was not only thrown to the wolves, but used to feed the wolves," Meghan Markle said in a promotional advertisement for the rings.
For his part, Prince Harry said, "They find it natural to lie to protect my brother (William), but they were not ready to tell the truth to protect us (Meghan and I)."
The first three episodes that the platform made available to its viewers since last Thursday were devoid of sudden scandals, as some had feared, as they focused on the beginning of the love story between the Duke and Duchess of Sussex until their marriage in 2018.
Follow our latest local and sports news and the latest political and economic developments via Google news


Midnight is a stunning black 12 year old Chow Chow that resembles a fluffy warm teddy bear. This handsome ebony boy is house broken, extremely dog friendly, good with cats, and adores people. He absolutely relishes at the opportunity to play, hug, and kiss. Very smart, Midnight knows when to voice his opinion and when to be stealth. He is completely healthy and the picture of perfect health.
Midnight was fortunate enough in his life to always have one person that loved him and went above and beyond for him. However, this is the second time he finds himself in desperate need of a home. His 'mom' moved into an apartment that did not allow dogs. Thus, she found him a home with a lovely elderly lady, where he lived many happy years. Then the time came when the elderly woman could no longer live on her own and was sent to a skilled nursing facility. She called Midnight's original mother, she found him a home in a boarding facility. Thus, for the past 2 months this senior doll has lived in a cage. It is highly unfortunate that this is what his life has come to. He was nothing but a loyal soldier to those that cared for him. Sadly, the boarding bill has become unaffordable to his original owner and she cannot keep him there any longer. Thus, she searched for a rescue.
Please let's focus on Midnight and refrain from making any comments that are not directly beneficial for him. We all feel his pain and want to help! Let's focus our energy on getting him a home and keep any negative comments regarding his 'humans' at bay.


text:
ISyE New Hires and Appointments
Apr 25, 2016 | Atlanta, GA
New Hires and Appointments
Georgia Tech's Stewart School of Industrial and Systems Engineering (ISyE) has hired new faculty in the last two semester. ISyE has also made several new faculty appointments, thanks to generous endowments from our alumni, as well as the College of Engineering.
George Lan joined ISyE in January 2016 as an Associate Professor. His research interests are in theory, algorithms, and applications of stochastic optimization and nonlinear programming. Most of his current work concerns the design of efficient algorithms with strong theoretical performance guarantees and superior practical performance for solving challenging optimization problem
Moe Trebuchon joined ISyE in August 2015 asEdenfield Executive-in-Residence. He is considered a subject matter leader in the application of Demand Forecasting, Replenishment, WMS, TMS, Order Management and Omni-Channel technology to the order fulfillment and delivery process having led a broad base of clients through complex technology enabled change programs.
Turgay Ayer was appointed the George Family Foundation Assistant Professor of Predictive Health in April 2016. He is also the research director for medical decision-making in the Center for Health & Humanitarian Systems at Georgia Tech. Dr. Ayer conducts research on stochastic modeling and optimization, with applications in predictive health, medical decision making, health care operations, and health policy.
David Goldberg was appointed A. Russell Chandler III Assistant Professor in February 2016. Dr. Goldberg works in applied probability, interpreted broadly, on topics ranging from inventory control and queueing theory to distributionally robust and combinatorial optimization. Much of his work focuses on using ideas from probability theory to prove that high-dimensional complex systems can be well-approximated by much simpler systems, and using these insights to devise novel algorithms with provable performance guarantees.
Sebastian Pokutta was appointed David M. McKenney Family Assistant Professor in April 2016. Dr. Pokutta's research concentrates on combinatorial optimization and polyhedral combinatorics, and in particular focuses on cutting-plane methods and extended formulations. His industry research interests are in optimization and machine learning in the context of analytics with a focus on real-world applications, both in established industries as well as in emerging technologies. Application areas include but are not limited to supply chain management, finance, cyber-physical systems, and predictive analytics.
Enlu Zhou was appointed Fouts Family Assistant Professor in May 2016. Her research interests include simulation optimization, stochastic control, and Monte Carlo methods, with applications in financial engineering and revenue management.
Nagi Gebraeel was named to a five-year appointment as Georgia Power Associate Professor in the College of Engineering in January 2016. Dr. Gebraeel currently serves as an associate director at Georgia Tech's Strategic Energy Institute with the responsibility of identifying and promoting research activities and thought leadership at the intersection of data science and energy. From the standpoint of application domains, he has a general interest in the energy industry with a focus on power generation, and the manufacturing industry with a focus on discrete and continuous manufacturing.
Turgay Ayer, George Family Foundation Assistant Professor of Predictive Health
David Goldberg, A. Russell Chandler Assistant Professor
Sebastian Pokutta, David M. McKenney Family Assistant Professor
Enlu Zhou, Fouts Family Assistant Professor
Nagi Gebraeel, Georgia Power Associate Professor, CoE
Shelley Wunder-Smith
Stewart School of Industrial and Systems Engineering


David is a Chartered Financial Planner and an Occupational Pension Specialist. He has worked in Financial Services for over 25 years and joined Attivo Financial Planning in 2017. David is a Fellow of the Personal Finance Society (PFS), part of the Chartered Insurance Institute (CII) and a Member of the Chartered Institute for Securities and Investment (CISI).
All Financial Planners require a Diploma in Financial Planning to provide advice, David holds both the Diploma and the Advanced Diploma in Financial Planning.


text:
Q: Angular how to ngFor the nested object properly? I have a JSON object like this in our API:
{
   "count": 58,
   "next": //API URL OF THE NEXT PAGE CUS WE HAVE PAGINATION HERE
   "previous": //API URL OF THE PREVIOUS PAGE
   "results": [
     {
      "game_id":3,
      "title":"Basketball"
     },
     {
      "game_id":4,
      "title":"Football"
     }
     //THE LIST GOES ON AND ON LIKE THIS
   ]
}

So I have a class like this;
export class TheGames{
    count:number;
    next:string;
    previous:string;
    public results = new class {
        game_id:number;
        title:string;
    }
}

In my ts file I subscribe them like this;
 game: TheGames[];
 //gameandcatservice is the service that I use and get games returns the games from API
  this.gameandcatservice.getGames().subscribe(data=>{
    this.game=data;

In html file I went like this;
<div *ngFor="let g of game['results']">
   <!-- SOMETHING HERE -->
</div>

I get the error of inside the console at browser;
ERROR TypeError: Cannot read property 'results' of undefined at....

I ont know why this happends because I have already defined results in the class and when I do this, I can acces to the elements inside results and print them to screen.
EDIT: Here is my getGames() function in service file;
  getGames():Observable<TheGames[]>{
    return this.http.get<theGames[]>(this.pathGames);
  }

Also when I console.log(this.games), I get  result like this;
    count: 58
    next: "http://localhost:8000/api/games?page=2"
    previous: null
    results: Array(10)
    0:
    game_id: 3
    title: "Basketball"
    __proto__: Object
    1:
    game_id: 8
    title: "Football"
    __proto__: Object
   //LIST GOES LIKE THIS FOR 10 ELEMENTS

A: Please change your class to
export class TheGames {
    count: number;
    next: string;
    previous: string;
    results: {
        game_id: number;
        title: string;
    }[];
}

Besides this, you assign this.game in an async manner. When the component is created this.game is undefined until the callback run. So you will need to handle this pending state.
One possibility would be to add the save navigation operator
<div *ngFor="let g of game?.results">
   <!-- SOMETHING HERE -->
</div>

A: You can solve the error by using ? safe navigation operator .The Angular safe navigation operator, ?, guards against null and undefined values in property paths
<div *ngFor="let g of game?.results">
   <!-- SOMETHING HERE -->
</div>

also you have a typo in your code, wrong class name theGames[] :
 getGames():Observable<TheGames[]>{
    return this.http.get<TheGames[]>(this.pathGames);
  }

here is a working stackbliz demo

A: Use an async pipe instead. Like so:
Component.ts
 game: this.gameandcatservice.getGames().pipe(
     take(1)
 )

Component.html
<div *ngFor="let g of (game | async)?.results">
   <!-- SOMETHING HERE -->
</div>

This will give you the added bonus of not having to unsubscribe from your observable if you would have needed to, and you won't have to initialise your variable, nor type it (it will infer its type from getGames on your service.)

redpajama_set_name: RedPajamaStackExchange


Some high end applications require multiple or critical calculations to be done by the microcontroller. This may lead to cases when the controller enters into wrong or infinite loops. As a result of this, the system either hangs up or gets crashed. The solution to overcome these situations is to automatically reset the system whenever such a situation arises.
The Watchdog Timer is a hardware or software generated timer interrupt which reboots/resets the system in the situations mentioned above. The watchdog timers are also used in cases when you intentionally require resetting the system without any physical interference.
The AVR microcontroller has an in-built watchdog timer. This article explains the working of watchdog timer in ATmega16.
The Watchdog Timer is a special timer which can be enabled in any section of the code and when enabled it ensures that a certain number of instructions execute within a pre-defined time frame. This time frame or the time delay can be configured/set using the registers of the watchdog timer. In case the instructions execute within the time frame, watchdog timer needs to be turned off and the program execution continues. However, if the instructions fail to execute within this time frame (this conditions is called time out condition) the entire system reboots thus avoiding any system crash or hang up.
The Watchdog timer of Atmega16 can be configured by using WDTCR register of AVR microcontroller. When the time out condition is set, the watchdog timer starts counting clock cycles. The watchdog's timer is clocked from separate on-chip watchdog oscillator of 1MHz frequency. The time out condition is set by configuring prescaler bits of WDTCR register.
WDTOE (Watch Dog Turn-Off Enable) – The watchdog timer is disabled by configuring WDTOE and WDE bits (explained later in the article).
WDE (Watch Dog Enable) – Watchdog timer is enabled by writing 1 to WDE bit.
1. Set the WDE and WDTOE bits in same clock cycle WDTCR register. The logic one must be written to WDE bit even though it is set to one already.
2. After four clock pulses, write logic 0 to the WDE bit. Otherwise watchdog timer will not be disabled.
To set watchdog timer condition at 2 seconds (approx.) and check the reset condition.
The connection for above mentioned objective is shown in circuit diagram. An LED is connected at pin PB0 which blinks continuously. Another LED is connected to PB1 pin. This LED will glow when watchdog time out condition occurs.
1. Check the status of WDRF bit. If this bit is high, set the PB1 pin of the controller.
2. Set the WDE bit in WDTCR register to activate the watchdog timer.
3. Set the prescaler bits WDP [2:0] = 111 for the time-out condition of 2 sec.
4. Write an infinite loop to toggle the PB0 bits toggles with a certain delay.
5. On time-out condition, watchdog timer resets the controller and this event reinitialize the program counter to go back at step 1.


text:
British bookings for Andalusian holidays are up this year, but Brexit casts a cloud of uncertainty
PEDRO LUIS GÓMEZ / PILAR MARTÍNEZ / RACHEL HAYNES
A fall in British tourist numbers after years of record-breaking figures, and Brexit just months away, meant that this week's World Travel Market (WTM) in London was going to be a challenging one for the Costa del Sol. The feeling, however, among
The unstoppable Damián Quintero fights for his first solo gold medal
MARINA RIVAS
...possible to move up the ladder towards the Olympics. So far he has begun well: he has earned a place in the final of the championship, which takes place this Saturday from 6pm, when he will be up against his most challenging rival. Quintero hasn't given ... and just one demanding duel faces him now. Nobody should expect to see him until then."I'm shutting myself away from the world until all this is over, focusing on my being and what lies ahead; he is a very challenging rival," he says. "My strategy has
Malaga surrender top spot after incident-packed loss to Osasuna
Daryl Finch
...following in. However, the tide would truly turn just five minutes later when Blanco Leschuk received a surprise second yellow for a high arm when challenging for an aerial dual just inside his own half. Out of options Malaga, by this point with only
A late-blooming passion leads to a call-up for the regional team
...park behind Estepona. It's beautiful, challenging and superb fitness training for us both." Travel costs However, like in any amateur sport, endurance riding success does come at a cost. While a number of other riders have sponsors, Caroline does it
Sergio García completes Andalucía Masters hat-trick after weather delay
ALEJANDRO DÍAZ
...reduced to 54 holes. "It's amazing to be able to win here at Valderrama three times - it's a dream come true," García said. "This golf course is so challenging and for me to be able to go out there in the conditions we played in all week and shoot three
Digging deep
Mark Nayler
...stage of a much more challenging task - namely, deciding what to do with the Valley of the Fallen once the dictator's remains are gone. The best suggestion, made by Spain's last Socialist government, was to turn it into a museum and education centre
Luis Enrique to lead Spain into new era as Fernando Hierro steps aside
Henry Flynn
challenging circumstances, Hierro told the Spanish football federation (RFEF) that he would be searching for new "professional challenges" having accepted responsibility for Spain's failure and turning down the opportunity to continue with Spain as the
Malaga's jábegas have arrived in Venice
BORJA GUTIÉRREZ
...that the event was not without its difficulties: "Spending hours in the sun without food or water was very challenging," he explaned. "Due to tiredness and poor organisation there were also a few crashes in the water. Near the end of the route a boat of
Mijas Rotary Club plants 100 trees to help restore stability to the natural world
Tony Bryant
...devastation and effects of deforestation worldwide by challenging students to raise funds to rebuild forests. The Rotary Club began the reforestation project in the Eco Reserva last year, when they planted 120 cherry and almond trees. "Planting a tree can
Changing place
THE EURO ZONE, MARK NAYLER
...political front lines; and the other involves managing one of the most challenging portfolios in European politics. On paper, at least, Escolano possesses the relevant experience. From 2000 to 2004 he was the chief economic advisor to then conservative


text:
Temperature may drop further tomorrow
UNB, Dhaka
Sat Jan 15, 2022 02:21 PM Last update on: Sat Jan 15, 2022 02:25 PM
Star file photo
Met office has predicted a 1-3 degree Celsius dip in temperature in the next 24 hours across the country.
Abul Kalam Mallik, a meteorologist with the Bangladesh Meteorological Department (BMD), confirmed it today.
According to the regular bulletin, "Weather may remain mainly dry with partly cloudy to cloudy sky over the country having chances of light rain or drizzle at one or two places over Khulna, Barishal, Chattogram, Dhaka and Sylhet divisions."
Moderate to thick fog may occur over the river basins and light to moderate fog may occur elsewhere over the country during midnight to morning, according to the bulletin.
The lowest temperature of the country was recorded at 8.9 degrees Celsius at Tetulia in Panchagarh on Saturday.


Minecraft : Revenge of the Wither is a game for Ouya and Nvidia Shield, based on the TV series of the same name. It's released in 2020 in most countries (Nippon being an exception).
Steve must explore several levels. He can attack with a enchanted Golden Sword and a Iron Axe. He can buy stronger weapons and armour at shops.
Steve has a health bar similarly styled to the Mega Man series. If it runs out, Steve falls off the screen in a similar fashion to Super Mario 3D Land/World. The health bar can be refilled by collecting Golden Apples.


Specifications / total length adjustment formula ## February price change 2016], steel case, single-tube, twin piston, rubber chamber type, ※: vehicle height adjustment range (standard vehicle ratio) F: + 3mm~-27mm R: -17mm~ -43mm / factory setting ride height (standard vehicle ratio) F: -20mm R: -30mm ※ touring selection and, in the case of solar panels with a car, will be re-used upper support, dust cover, the bump rubber. In addition, other vehicle separately, you will need the following genuine parts. Front suspension support SUB-Assy (part number: 48609-47030) two / strut mounting bearing (part number: 48619-28010) two / front coil spring seat upper (part number: 48471-42010) two / front coil spring insulator upper (part number: 48157-47020) two / front spring bumper (part number: 48331-75010) you will need to shorten the processing of two ※ front spring bumper.
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