You may be thinking to rank for different keywords, but may not be thinking about the competition. Some keywords can be very hard to rank just because some of the top blogs may be also trying to rank for the same. It is that's why important that you should try to rank for keywords which are not very competitive. You should try to rank for competitive keywords only if you have been easily able to rank for keywords having less and medium competition.
According to me, one should first start blogging and then think about keywords ranking in future. If you'll from the start think about ranking for different keywords, then you may never get success.
I see many newbies making the mistake of not having proper title for their blog posts. Please remember that you won't be able to rank for keywords or may not be able to maintain the ranking if your blog post's title is not a proper one.
You should always try to include the keyword in the blog post title. If you won't have the keyword in your blog post title, then achieving good search engine ranking can be very hard. At the same time you should try to have nothing more than 60 characters in your blog post title.
Very long titles are of no use and that's why I'll recommend you to avoid them completely.
Always try to have proper description for all your blog posts. I have seen that my blog posts which have proper description rank better than blog posts which don't have proper descrition.
A proper description is the one which includes not more than 160 characters and at the same time includes the keywords.
Your blog post description can be a very short summary about the blog post. Always try to keep the descrition as appropriate as possible as people may tend to land on your blog post just because of the appropriate descrition that you have used for your blog post.
You can't expect your blog posts to rank if the quality is not good or if the content length is short.I have seen that posts with more than 1500/2000 words rank much easily than posts having 500 or 800 words only. The main benefit of writing lengthy posts is that you'll be to maintain the keywords ranking for a longer duration.
If you'll regularly post good quality posts with minimum of 1000 to 1500 words, then competitors may not be able to outrank you easily. You need to plan your posts, do proper keyword research and at the same time you need to keep improving the quality of content as it'll be helpful in attracting more readers easily.
If you're looking forward to really rank your blog posts, then you shouldn't miss out on making use of 'ALT Tags'. You should always try to make use of keyword in the ALT tags. As this can be definitely helpful in getting better keywords ranking. Many bloggers miss out on making use of ALT tags which can definitely be one of the reasons that they may not be able to get good search engine rankings.
Newbies think that internal linking is not very beneficial, but they're wrong.
Internal linking is helpful as you'll be able to get back-links from your own blog. As well as internal linking can help in getting better Alexa ranking. The day from which I started focusing on doing internal linking properly. It helped me in improving the bounce rate and at the same time I was able to get better ranking for various keywords.
While doing internal linking, always try to interlink related posts only. At the same time you should avoid interlinking to many old posts from a single blog post as the readers may find it boring. Too much internal linking can even harm your keywords ranking.
Back-links are very important as they will help you in getting better keywords ranking.
Commenting and guest posting on other blogs related to the same niche will provide your blog with good backlinks which will improve your keywords ranking.
When it comes to commenting you should always try to comment at related blogs only. You should do commenting regularly, but excessive commenting should be avoided as it may result in Google penalty.
I'll advice you to always focus on natural link building as Google is against artificial and un-natural link building practices.
In a previous post I made my blog readers know the tips to double social media traffic as social media marketing is very important.
If you want to get better keywords ranking in Google search engine. Then you need to get more number of social shares.
You need to have your presence at Facebook, Twitter, Google Plus, LinkedIn and other social networking sites.
For getting more number of social shares you need to share your posts at the right time. At the same time you need to adopt the best social media marketing strategies for getting maximum number of social shares.
There are many bloggers who have got lot of success. Just because of the reason that they have decided to target long-tail keywords only.
Ranking for long-tail keywords is easier than ranking for two to three words keywords having lot of competition.
You can make use of Long Tail Pro tool for finding out different long tail keywords. This tool has helped many bloggers in getting lot of traffic by targeting variety of long tail keywords.
I hope these valuable tips will help you out in getting the best keywords ranking for all your blog posts. Or other websites you are trying to improve. Dedication, determination and confidence is what you need to have for achieving the Google keywords ranking. And it's a regular practice which delivered the result.


text:
On March 4, 1958, under the leadership of Jawaharlal Nehru, for the first time in the history of independent India, Parliament passed a resolution on science policy. The resolution stated: "Science... has provided new tools of thought and has extended man's mental horizon. It has thus influenced even the basic values of life, and given to civilization a new vitality and a new dynamism." The resolution said the aim of the scientific policy was, among other things, to "encourage individual initiative for the acquisition and dissemination of knowledge, and for the discovery of new knowledge, in an atmosphere of academic freedom." In effect, this resolution proved to be a springboard for the development of the country's scientific infrastructure. Since that resolution, successive governments issued policy statements with varying emphasis on chosen objectives and goals, often echoing the existing national and global imperatives and the ruling dispensation's ideology.
India's Department of Science and Technology recently released a draft of the fifth Science, Technology, and Innovation Policy. This 62-page-long document presents the objectives and goals of our new science policy. The public is expected to provide feedback on this document before it gets finalised. But the problem with the document is that it is rambling and full of jargon and clichés, making the task of separating the grain from the chaff a major exercise in itself.
Also read | Science policy to help researchers access top journals for free
The new policy envisages technological self-reliance and aims to position India among the top three scientific superpowers. Though the names of the other two nations are not mentioned, it must be understood that they are the U.S. and China. For that to happen, the draft policy says, we need to attract our best minds to remain in India by developing a "people-centric" science, technology, and innovation "ecosystem". It states that the private sector's contribution to the Gross Domestic Expenditure on Research and Development should be doubled every five years. The 2013 policy had similar aims. The 2020 draft policy fails to discuss what we have achieved on these fronts since then.
Why does our R&D investment in science continue to hover between 0.5% and 0.6% of the GDP? Raising it to 2% of the GDP has been a national goal for a while. Despite strong recommendations in the past by several scientific bodies and leading scientists and policymakers, we are still well short of that goal. The 2020 draft policy blames this on "inadequate private sector investment" and adds that "a robust cohesive financial landscape remains at the core of creating an STI-driven Atmanirbhar Bharat." It looks as if the government is trying to shift the responsibility of financing R&D to different agencies such as the States, private enterprises, and foreign multinational companies. But it is doubtful if the various funding models that are presented are workable or practical, especially during a pandemic.
The policymakers who drafted this report should have gone back to the self-financing revenue model proposed in the Dehradun Declaration for the CSIR labs back in 2015 and critically evaluated its success rate. Common sense informs us that the private sector cannot be expected to pay for basic research. This is because the return on investment in basic research takes too long from a private sector perspective. Only the government can have long-term interest to support such research. Participation of the private sector in basic science has not happened even in the U.S. The fact is that basic science research in India is suffering from the lack of adequate funding despite grand proclamations. Even elite institutes like the Indian Institutes of Technology are finding it difficult to run their laboratories on a day-to-day basis because of paucity of funds.
Comment | Academic research is necessary, but not sufficient
The draft policy visualises "a decentralized institutional mechanism balancing top-down and bottom-up approaches, focusing on administrative and financial management, research governance, data and regulatory frameworks and system interconnectedness, for a robust STI Governance". This is easier said than done. This intention is in fact defeated in the document itself, where several new authorities, observatories and centres have been proposed, which may end up feeding up the already fattened bureaucracy in science administration. Decentralisation of administrative architecture is essential, but we need to explore the practical option of providing more autonomy to research and academic centres for financial management.
Some points are welcome. These include the fact that policymakers are considering alternative mechanisms of governance of the financial landscape; that they realise the administrative burdens of researchers and the problem of journal paywalls; and promise to explore international best practices of grant management.
It was reported in 2019 that more than 2,400 students dropped out from the 23 IITs in just two years, with over half of them belonging to the Scheduled Caste/Scheduled Tribe and Other Backward Classes. The number of suicides of students is also on the increase in the IITs. Caste discrimination could be one of the reasons for these tendencies. As a part of inculcating an inclusive culture in academia, the document promises to tackle discriminations "based on gender, caste, religion, geography, language, disability and other exclusions and inequalities". It mentions more representation of women and the LGBTQ community, but is silent on how we are to achieve their proportionate representation.
Also read | Situation in IIT Madras grave when it comes to SC/ST students and faculty, says NCSC member Swaraj Vidwaan
In the chapter 'Science Communication and Public Enagagement', concerns on the disconnect between science and society are valid. But the fact is that hyper-nationalism is not conducive to the propagation of evidence-based science and a rational outlook. It is also heartening to see that the document harks back to our constitutional obligation to "develop a scientific temper, humanism and the spirit of inquiry and reform." But it is silent on how this can be achieved when pseudoscience is deliberately propagated in the name of traditional science with the help of some arms of the government. A recent instance is the proposal by the Rashtriya Kamdhenu Aayog to conduct a national examination under the garb of 'cow science'.
Comment | Pallbearers of 'cow science'
The document does not mention how to stem the rot within, although it speaks extensively about science communication and scientific temperament. Our belief systems, values, and attitudes have an impact on the quality of research. That partly explains why Indians who have chosen to work in labs abroad are able to make path-breaking discoveries. The ruling dispensation has a moral obligation to facilitate an environment that encourages a mindset that constantly challenges conventional wisdom as well as open-minded inquiry among the students. Only a dissenting mind can think out of the box.
The document contains nuggets of modern scientific vision and information. But its digressive style impacts the reader's attention. The document should prioritise important issues and amplify first the problems which have cultural and administrative dimensions. With the advent of new disruptive technologies, global competitiveness will be increasingly determined by the quality of science and technology, which in turn will depend on raising the standard of Indian research/education centres and on the volume of R&D spending. India has no time to waste.
C.P. Rajendran is an adjunct professor in the National Institute of Advanced Studies, Bengaluru. Views expressed are personal
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redpajama_set_name: RedPajamaCommonCrawl


text:
Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection
Sriram Bandi1, 2 and
Ramesh Akkina1Email author
© Bandi and Akkina; licensee BioMed Central Ltd. 2008
Received: 31 October 2007
Human embryonic stem (hES) cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC) arise from CD34+ hematopoietic progenitor cells (HPCs) and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection.
Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC) were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC). The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs.
Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.
Green Fluorescent Protein Expression
Hematopoietic Progenitor Cell
Antigen Uptake
Mixed Leukocyte Reaction
Mouse Bone Marrow Stromal Cell
Human embryonic stem (hES) cells are endowed with pluripotential and self-renewal properties [1, 1]. In addition, they are tractable for stable genetic modification. These attributes qualify them as potential candidates to derive an unlimited supply of any cell type for transplantation, gene therapy, drug screening and functional genomic applications. A number of previous studies have demonstrated the ability of hES cells to differentiate into a myriad of cell types that include neurons, hematopoietic cells, cardiomyocytes, and insulin-secreting cells, to name a few [3–9]. Many new studies are currently directed towards expanding the use of hES cells for novel applications.
In this regard, the ability to generate cells of the hematopoietic system has considerable potential in several areas of clinical and experimental medicine as they can reconstitute the entire blood system and can serve as primary targets in gene therapy in treating infectious diseases such as AIDS and inherited diseases [9, 10]. Given the present lack of effective vaccines and the ineffectiveness of drug based therapies for a complete cure with regard to HIV/AIDS, new and innovative approaches are essential [10, 11]. Gene therapy through intracellular immunization offers a promising alternative approach and possible supplement to current HAART therapy. A primary goal of many ongoing studies is to introduce an effective anti-HIV gene into hematopoietic progenitor cells [11]. As these cells possess the ability to self-renew, they have the potential to continually produce HIV resistant T cells, macrophages, and dendritic cells in the body thus providing long term immune reconstitution. These approaches use CD34+ hematopoietic stem cells for anti-HIV gene transduction via integrating viral vectors such as lentiviral vectors. Current sources of CD34+ cells are restricted to human umbilical cord blood (CB), adult bone marrow (BM), mobilized peripheral blood, (MPB), and fetal liver [11]. hES cells are a good viable alternative for the generation of an unlimited supply of CD34+ cells thus paving the way for utilization of these cells for hematopoietic cell therapy [9]. Recently we demonstrated derivation of phenotypically and functionally normal macrophages from hES-CD34+ cells and established that they could support HIV-1 infection. These studies laid the ground work for utilizing hES-CD34+ cells in HIV research and for testing anti-HIV genes in a gene/cell therapy setting [10].
Similar to monocytes/macrophages, dendritic cells (DCs) also originate from hematopoietic progenitor cells and spread via the bloodstream and lymphatics [12, 13]. They are found in almost every organ as sentinels of the immune system. In innate immunity, DCs function via type-1 interferon activation of both macrophges and NK cells. In adaptive immunity, DCs constitute the most powerful antigen presenting cells (APCs) that prime naïve T lymphocytes and sensitize cytotoxic T lymphocytes to the antigens they present [13]. Thus, efficient generation of these cells from renewable sources such as hES cells would have great potential for immunotherapy applications. However in HIV-1 infection, in addition to being infected and functionally compromised, paradoxically they are also culprits in the efficient transfer of the virus to susceptible cells [14]. Thus in gene therapy applications for HIV infections they are also among principal cells that need to be protected. For such efforts to proceed further, it is important to evaluate if hES derived DCs are functionally normal and support HIV-1 infection. As a first step towards this goal, here we show that hES-CD34+ cells can give rise to normal DCs which are capable of supporting HIV-1 infection.
Hematopoietic differentiation of human ES cells and derivation of dendritic cells
hES cell line H1 was propagated as undifferentiated cells by co-culture on mitomycin treated MEF feeder layers. Consistent with previous studies, cells cultured in this manner grew as tightly packed colonies (Figure 1A). To promote hematopoietic differentiation, the H1 cell colonies were cocultured with irradiated S17 cells [10]. After 4–7 days in culture, the H1 cells differentiated into cystic bodies (Figure 1B) which were allowed to further expand for 14–17 days. FACS analysis of single cell suspensions of the differentiated cells showed the development of CD34+ cells (range 7% to 15%, data not shown). Purified CD34+ cells were later cultured in dendritic cell differentiation media in parallel with CD34+ cells from fetal liver. Cell morphology and phenotypic properties were monitored periodically. By day 12, the differentiating cells showed a characteristic veiled or dendritic appearance with numerous cytoplasmic extensions indicative of DC development (Figure 1C). FL-DCs and hES-DCs were found to be morphologically similar as seen in the phase-contrast images (Fig 1C and 1D). We also looked for the expression markers during differentiation and figure 2 illustrates CD1a and CD14 expression at days 0, 3, and 12. At day 0, CD34+ cells expressed neither CD1a nor CD14. By day 3, two cell populations expressing either CD1a or CD14 could be seen with the CD14 single positive population being the majority. With hES CD34+ cells, by day 12, 34% of the cells differentiated into CD1a expressing DCs. The overall yield of CD1a+ cells prepared in this manner ranged from 8–34% for hESC-DCs and 20–64% for FL-DCs.
Derivation of DCs from hES cells: Undifferentiated hES cells were cocultured with S17 mouse stromal cells to derive cystic bodies. Later, purified CD34+ cells derived from cystic bodies and fetal liver were cultured in cytokine media to derive DCs as described in Methods. A and B, representative hES colony and an cystic body respectively. C and D, morphology of DCs differentiated from hES and FL derived CD34+ cells.
FACS analysis of differentiating DCs from hES and FL CD34+ cells: CD34+ cells were cultured in cytokine media and analyzed by FACS for CD14 and CD1a markers at different days by staining with CD1a-PECY5 and CD14-PE conjugated antibodies. Dot plots are representative of triplicate experiments.
hES derived DCs (hES-DC) express normal DC surface markers
The mature DCs were generated from hES cells by culturing for 12 days in media containing the growth factors and cytokines as mentioned above. Following differentiation, hES-DCs were analyzed for expression of the human class II antigen presenting molecule HLA-DR and the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) by FACS. The antigen presenting cell surface marker HLA-DR present on mature DCs is critical for presenting antigen to T cells and the co-stimulatory molecules B7.1 and B7.2 activate T cells [13–17]. For a two color FACS analysis, cells were stained with CD1a and HLA-DR, CD1a and B7.1, and CD1a and B7.2. Results showed that hES derived DCs are positive for HLA-DR, B7.1, and B7.2 surface expression (Figure 3). The expression levels are comparable between hES-DCs and FL-DCs: CD1a+HLA-DR+ (10.5% and 9.6%), CD1a+B7.1+ (14% and 15.4%), and CD1a+B7.2+ (11.4% and 12.9%) cells. We also observed single positive cell populations for CD1a, HLA-DR, B7.1, and B7.2 which most likely represent other subsets of DCs. These above data indicate that phenotypically normal DCs can be generated from hES-CD34+ cells.
Phenotypic analysis of hES-DCs and FL-DCs: hES-DCs and FL-DCs were stained with antibodies CD1a-PECY5, HLA-DR-PE, B7.1-PE, B7.2-PE, and DC-SIGN-PE. Expression of these respective markers was analyzed by FACS. Percent positive cells are indicated in respective plots for each of the cell surface markers. The isotype controls are shown in the left panel. Data is representative of triplicate experiments.
hES-DC express Dendritic Cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN)
DC-SIGN (CD209) is a DC-specific C-type lectin which is expressed by mature DCs. DC-SIGN plays a vital role in establishing the initial contact between DCs and resting T cells through its recognition of ICAM-3 receptor [13–15]. In addition, with regard to HIV infection, it was found that DC-SIGN bound virus is more stable and is more efficiently transferred to susceptible target cells [13, 14]. We therefore examined the hES-DCs for its presence by staining with anti-DC-SIGN-PE and anti-CD1a-PECY5 conjugated antibodies. Results showed that a significant percentage of hES-DC (14.4%) express DC-SIGN similar to FL-DCs (14.3%) (Figure 3). We also observed cell populations single positive for CD1a and DC-SIGN which could represent other DC subtypes.
hES-DCs are capable of allogeneic T cell stimulation
Since DCs are capable of potent stimulation and proliferation of allogeneic T cells, we sought to determine whether the hES-DCs were also able to elicit such a response in a mixed leukocyte reaction (MLR). The day 12 differentiated hES-DCs and FL-DCs, immuno-magnetically sorted based on CD1a+ were used, and proliferation was measured by BrdU uptake using FACS after staining with PE-conjugated anti-BrdU antibody as described in Methods. Results showed that hES-DCs mediated a significant stimulation of allogeneic T cells similar to FL-DCs (Figure 4). The ratio of DCs to the T cells in the reaction mix is expressed as 1:125, 1:250, 1:500, 1:1000, 1:2000 and 1:4000. Highest stimulation was seen with the lower DC to T cell ratio (1:125) and the levels of stimulation decreased at higher ratios. The percent stimulation of T-cells by hES-DCs and FL-DCs were similar at the ratios 1:500 to 1:4000. These results demonstrated the capacity of hES-DCs for allogeneic T cell stimulation.
Allogeneic T-cell stimulation by hES and FL derived DCs: The allogeneic stimulatory properties of DCs were assessed in a mixed leukocyte reaction assay using allogeneic T-cells. Graded numbers of sorted and irradiated DCs were co-cultured with 5 × 105 allogeneic T cells. BrdU incorporation was determined by FACS using a PE-conjugated antibody against BrdU. Histograms depict relative percent of BrdU uptake when compared to positive control cells stimulated with IL-2 and PHA. The X-axis is expressed as ratio of stimulator DCs cells to allogeneic responder T cells.
hES-DCs are capable of antigen uptake
An essential function of the DCs is their ability to capture and present antigen to T-cells. The capacity of DCs to take up antigens was measured by using Alexa-dextran, as an indicator of mannose-receptor (MR)-mediated endocytosis. The level of antigen uptake by DCs was expressed as the difference in percentages between the test samples incubated at 37°C versus the controls at 0°C. Results showed that hES-DCs are capable of antigen uptake. However, the levels of uptake were found to be about one fold less than that of FL-DCs (27.5% versus 61.0%) (Figure 5). This could be due to differences in cell types of origin and/or due to their physiological condition at the time of harvest. These results further support the notion that the hES-DCs are functionally competent in addition to being morphologically and phenotypically normal.
Antigen uptake by hES-DCs: Cultured hES and FL DCs were sorted based on CD1a marker. The cells were then incubated with Alexa-Dextran at 0°C and 37°C for 1 hr and analyzed by FACS as described in Methods. The percent antigen uptake was measured as the difference in percentages between the test (37°C) and control (0°C). The percent positive cells are indicated in the plots for both hES-DCs and FL-DCs. Data are representative of triplicate experiments.
hES-DCs can support productive HIV-1 infection
The above results have collectively shown that hES-DCs are similar to normal DCs as demonstrated by comparative analysis with FL-DCs. Apart from being critical for host immunity, DCs can be infected and disabled by viruses such as HIV-1 [13, 14]. As mentioned above, DCs also play an important role in the natural history of HIV infection. At the early phase of HIV-1 transmission, DCs capture HIV-1 at mucosal surfaces and transmit the virus to T-cells in the vicinity. Capture of the virus on DCs is known to take place via C-type lectin DC-SIGN surface molecule. Therefore, we wanted to determine if hES-DCs were susceptible to HIV-1 infection as compared to normal DCs. Accordingly, the CD1a-sorted hES-DCs and FL-DCs were exposed to HIV-GFP, a T-tropic virus containing the gene for green fluorescent protein (GFP). Our results showed that both of the virus-exposed hES-DCs and FL-DCs supported viral infection based on GFP expression by the respective cells. However, not all the cells in the culture were productively infected as only a fraction of cells were virus positive for GFP expression. (Figure 6). Supernatants from infected DC cultures were also positive of p24 viral antigen indicative of productive infection. The levels of virus production were not copious however, which is not unexpected since DCs are known to support only a low level viral replication [13, 18].
HIV-1 infection of hES and FL DCs: To determine virus susceptibility, FL and hES DCs were infected with a replication competent X4-tropic HIV-GFP reporter virus strain at an m.o.i. of 0.2. At 6 days post infection, cells were visualized by fluorescence microscopy to determine GFP expression in productively infected cells at the single cell level. Phase contrast and fluorescence images are shown for the respective cell types (A). Infected culture supernatants were assayed for viral p24 antigen by ELISA at different days post-infection (B). Data is representative of duplicate experiments.
Towards the goal of exploiting hES cells for novel hematopoietic cell reconstitution and HIV gene therapies, here we have shown that phenotypically normal and functionally competent dendritic cells could be differentiated from hES-CD34+ cells. Moreover, we also have demonstrated for the first time that hES-DCs can be productively infected with HIV-1 thus allowing future testing of anti-HIV therapeutic genes such as siRNAs for efficacy in these cells.
In these studies, we induced hES derived CD34+ cells to differentiate into myeloid DCs in the presence of cytokines SCF, GM-CSF, Flt3, IL-3, TNF-α, and IL-4. CD34+ cells derived from human fetal liver were also evaluated in parallel for comparison. Based on FACS analysis for surface markers during culture, CD34+ cells differentiated into mature myeloid DCs showing the typical CD1a phenotype similar to those derived from FL CD34+ cells. The morphology and phenotypic characteristics of hES-DCs were found to be similar to that of DCs derived from fetal liver CD34+ cells cultured in parallel.
It is important that hES-DCs are also functionally normal for future applications. Therefore we analyzed the functional markers HLA-DR (MHC-II), B7.1 (CD80), and B7.2 (CD86) typically expressed by mature DCs. The antigen presenting cell surface marker, HLA-DR present on mature DCs is critical for antigen presentation to CD4+ T cells and the co-stimulatory molecules B7.1 and B7.2 are needed to activate T cells. The mature hES-DCs expressed HLA-DR, B7.1, and B7.2 surface molecules which were comparable with levels expressed in FL-DCs. Consistent with the DCs' ability, the hES-DCs also showed normal capacity for antigen capture as evidenced by dextran uptake. We further analyzed the capacity of hES-DCs to induce proliferation of allogeneic T cells in a mixed leukocyte reaction. Our results showed that hES-DCs indeed are capable of mediating this allogeneic response. We also evaluated the expression of another cell surface molecule, DC-SIGN (CD209) which is a DC-specific adhesion receptor belonging to the C-type lectin family involved in the interactions with T cells [13, 14]. Our results showed similar levels of DC-SIGN in hES-DCs and FL-DCs. The above data taken together showed that hES-DCs are phenotypically and functionally normal.
It is known that HIV-1 can infect DCs with the virus remaining stable for long periods. DCs transmit the virus efficiently to CD4 T cells and therefore play an important role in HIV-1 infection. Our results showed that hES-DCs were susceptible to HIV-1 infection similar to that of FL-DCs. However, not all the cells in the culture were productively infected and the levels of viral production are low. This is consistent with previous findings that DCs support only a low level replication and fully mature DCs may have a block in viral replication [13, 14]. A recent report has also demonstrated the derivation of physiologically normal DCs from hES-CD34+ cells using a different protocol and compared these to those derived from adult human CD34+ cells and peripheral blood monocytes [19]. Our results are in agreement and confirmed these previous findings, and additionally extended them further by demonstrating that hES-DCs are also susceptible to HIV-1 infection.
In HIV-1 disease, infection of CD4 T cells leads to their eventual decline whereas infection of monocytes/macrophages and dendritic cells leads to continued viral spread and defects in antigenic presentation thus exacerbating the disease process [14]. We previously demonstrated the derivation of macrophagres from hES-CD34+ cells whereas studies of Galic et al [20] derived functional T cells from hES-CD34+ cells in vivo using humanized mice. Whether the hES derived T cells support HIV-1 infection remains to be determined. Lentiviral vector transduction of hES cells and derivation of functional macrophages and T cells that retained the expression of the transgene established that hES cells are tractable for deriving gene modified end-stage primary hematopoietic cells [10, 20]. Moreover, our present results together with our previous findings that both hES derived DCs and macrophages are susceptible to HIV infection paved the way for testing anti-HIV constructs introduced into either hES cells or their derivative hematopoietic progenitor CD34+ cells. Thus far many previous studies including our own evaluated a variety of anti-HIV gene constructs in a hematopoietic stem cell setting using CD34+ cells from routine sources such as bone marrow and cord blood [11]. Newer and more potent novel constructs such as siRNAs are currently being investigated some of which are currently entering clinical trials [21]. In addition to anti-HIV genes with a direct inhibitory action on viral molecules, siRNAs and ribozymes that down regulate cellular molecules that aid in HIV-1 infection such as viral coreceptors CCR5 and CXCR4 also show considerable promise [22, 23]. Such constructs can now be introduced into hES cells and their efficacy tested in end-stage cells represented by DCs, macrophages, and T cells.
In summary, our data demonstrated the development of terminally differentiated DCs derived from hES cells. The hES-DCs display typical DC morphology, express normal phenotypical markers, are capable of antigenic stimulation, and support HIV-1 infection.
Phenotypically normal and functionally competent dendritic cells could be derived from hES-CD34+ cells. Large numbers of these hES-DCs cells can now be cultured from a renewable source for use in cell and immune-based therapies. Since these cells also support productive HIV-1 infection, they provide a uniform source of DCs for viral infection studies. It is also now feasible to gene transduce either hES cells themselves and/or hES derived CD34+ cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated DCs which are among the primary target cells that need to be protected against HIV-1 infection.
hES cell growth and propagation
H1 human embryonic stem cell line (hES) was obtained from WiCell (Madison, Wisconsin). The undifferentiated cells were maintained by co-culture with mitomycin C treated mouse embryonic fibroblast (MEF) cells (Chemicon, Temecula, CA) in DMEM/F12 medium supplemented with 20% knockout serum replacer (Invitrogen), 1% MEM-non essential amino acids (Invitrogen), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen), 0.5% penicillin/streptomycin and 4 ng/ml human basic fibroblast growth factor (Invitrogen). Culture media was replaced daily with fresh complete medium. Mature colonies were subculture weekly by digesting with collagenase IV (Invitrogen) as previously described [10].
Differentiation of hES cells into DCs
The undifferentiated hES cells (H1) were harvested by treatment with 1 mg/ml collagenase IV (Invitrogen) and dispersed by scraping to maintain the cells in small clumps. The hES cells were added to irradiated (35 Gy) S17 mouse bone marrow derived cell layers and cultured with differentiation media composed of RPMI supplemented with 15% FBS (HyClone), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% MEM-nonessential amino acids, 1% penicillin/streptomycin. Media was changed every 2 to 3 days. After indicated days (14–17 days), the differentiated hES cystic bodies were harvested and digested into single cell suspension using collagenase type IV followed by 0.05% trypsin/EDTA supplemented with 2% chick serum (Invitrogen) for 20 minutes at 37°C. Cells were washed twice with phosphate-buffered saline (PBS), filtered through a 70-μM cell strainer (BD Biosciences). To assess the levels of CD34+ hematopoietic progenitor cells in the bulk cell suspension, cells were labeled with PE conjugated anti-CD34+ antibody (BD Biosciences, San Jose, CA) and analyzed by FACS. To purify the CD34+ cells, Direct CD34+ Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA) was used as recommended by the manufacturer's protocol. Isolated CD34+ cell purity was determined by FACS like above. For comparative experiments, human CD34+ cells were also purified from fetal liver tissue as described above [24]. To derive DCs, the purified CD34+ cells (~4 × 105 to 6 × 105 cells) were cultured in Iscove's media containing 10 ng/ml each of SCF, IL-3, TNF-α, IL-4 and 50 ng/ml each of GM-CSF and Flt-3. The differentiated mature dendritic cells were used for subsequent phenotypic and functional analysis.
Phenotypic analysis of hES-DCs
To determine if hES derived DCs were phenotypically normal, analysis of the characteristic cell surface markers was performed by FACS using respective conjugated antibodies against CD1a-PECY5, CD14-PE, HLA-DR-PE, B7.1-PE, B7.2-PE and DC-SIGN-PE. Fetal liver CD34+ cell derived DCs were also evaluated in parallel. Blocking step was first performed by incubating the cells with the respective isotype sera control for 30 minutes at 4°C before staining with the respective cell surface marker antibodies. Isotype control staining was used to determine background levels. FACS analysis was performed on Beckman-Coulter EPICS®XL-MCL flow cytometer with data analysis using EXPO 32 ADC software (Coulter Corporation, Miami, FL). A minimum of 10,000 cells were analyzed in each FACS evaluation.
Functional analysis of hES-DCs by Mixed Leukocyte Reaction (MLR) assay and antigen uptake assay
The T cell stimulatory capacity of DCs derived from hES cells CD34+ progenitor cells was assessed by co-incubating graded numbers of CD1a+ cells previously sorted on the basis of CD1a immunomagnetic labeling (Miltenyi Biotech, Auburn, CA), and irradiated (3500 rads) DCs for 5 days with 5 × 105 allogeneic peripheral T cells isolated from peripheral blood using a column purification method to isolate resting T cells per manufacturer's instruction (Cedarlane, Ontario, CA). BrdU (10 μM final concentration) was added 18 hr before harvest and incorporation was measured by permeabilizing the cells with ice cold 70% ethanol for 20 min followed by washing in PBS. The cells were resuspended in freshly prepared 2 N HCl and incubated for 20 min at room temperature to denature nuclear DNA. The cells were then neutralized with 0.2 M disodiumborate and washed with PBS twice. Cells were stained for 20 min with anti-BrdU antibody conjugated with PE (BD-Pharmingen, San Jose, CA). Cells were washed with PBS and analyzed by FACS to determine the percent incorporation of BrdU which is indicative of proliferation. The percent BrdU was determined as a function of input number of sorted DCs and plotted as percent BrdU staining vs. input numbers of DCs. Alexa-dextran was used to assess cell endocytosis as previously described [25]. The antigen uptake capacity was determined using CD1a+ immunomagnetic purified hES-DCs and FL-DCs. Cells resuspended in 10% FBS Iscove's medium (~1 × 105 cells) were incubated with 1 mg/ml Alexa-dextran at 37°C and 0°C for 60 minutes. The cells were later washed with PBS five times prior to FACS analysis. The level of antigen uptake by DCs was expressed as the difference in percentages between the test (37°C) and control samples (0°C). Fetal liver derived CD34+ cells were also evaluated in parallel.
HIV-1 infection of hES cells derived dendritic cells
To determine if hES-DCs can be infected with HIV-1 and support viral replication, cells were incubated with a X4 tropic replication competent HIV-GFP reporter virus NLENG-IRES [10, 26]. An m.o.i of 0.2 in the presence of 4 μg/ml polybrene was used. Infected cells were visualized by fluorescence microscopy to identify GFP expressing cells. Infected culture supernatants were also assayed for p24 antigen by ELISA using a Coulter-p24 kit (Beckman Coulter, Fullerton, CA).
Work reported here was supported by NIH RO1 grants AI50492 and AI057066 to R.A. We thank Joseph Anderson for suggestions, William Wheat for help with MLR and antigen uptake assays, Sarah Akkina and Jennifer Quick for help with maintaining hES cells and culturing cystic bodies. We thank Leila Remling for isolating fetal CD34+ cells, and the NIH AIDS Research and Reference Reagents Program for HIV-1 related reagents used in this work.
The author(s) declare that they have no competing interests.
SB derived the experimental data and RA was responsible for the conception and overall implementation of the project. All authors read and approved the final manuscript.
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, 80523, USA
Department of Medicine, Marion Bessin Liver Research Center and Center for Human Embryonic Stem Cell Research, Albert Einstein College of Medicine, Bronx, New York, 10461, USA
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Public affairs is more than lobbying
Public affairs is more than lobbying What do banking lobbyists do in Bern? And which issues are at the top of this year´s political agenda? https://www.swissbanking.org/en/services/insight/1.15/public-affairs-is-more-than-lobbying https://www.swissbanking.org/en/services/insight/1.15/public-affairs-is-more-than-lobbying/@@download/image/115 (9).jpg
The serious consequences of the negative interest rates
Der neue Leiter Finanzmarkt Schweiz im Interview
What do banking lobbyists do in Bern? And which issues are at the top of this year´s political agenda?
The term "lobbying" is not broad enough to describe the jobs of Kuno Hämisegger, Delegate of Public Affairs and Alena Weibel, Head of Political Issues. The two Swiss Bankers Association´s employees represent the interests of the Swiss financial centre at the federal parliament building. Their job is a "two-way road", as Kuno Hämisegger fittingly describes it. They act as the link between the bankers association and policymakers, introducing the issues faced by the financial centre into the political decision making process as early on as possible, and overseeing these until they are implemented. This is no easy task, as the financial centre has had a major impact on the political agenda since the outbreak of the financial crisis. In 2015 there are once again a number of key projects to be discussed and decided on, for example the automatic exchange of information and double taxation agreements, or the switch from the current withholding tax scheme to a paying agent tax.
Kuno Hämisegger and Alena Weibel share their views in the Bankerscast, a Podcast dedicated to the Swiss financial centre and its banking regulations from the Association of Foreign Banks in Switzerland.
Von Geschäftsmodellen, Strategien und Standards
France Régularisation des avoirs non conformes fiscalement
by Jakob Schaad, Former Head of Private Banking & Asset Management and Deputy CEO
Previous: The serious consequences of the negative interest rates
Next: Looking to the future with optimism
Céline Freivogel
Head of publications


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John Wall Street
Endeavor Buying On Location Experiences at +/- 12x EBITDA, NFL to Retain Stake
JohnWallStreet
It has been reported that Endeavor is closing in on a deal to acquire the 80% of On Location Experiences (OLE) not owned by the NFL for between $650 million and $700 million; the league plans to retain their 20% stake in the company. The NFL's ongoing participation - and the 9,500 Super Bowl tickets/year that OLE gets from the league at face value (+ PrimeSport's allotment) - is critical to the business' success; the league was responsible for generating 40% of the company's total revenue last year (balance: 40% other sports, 20% music & travel). In 2018, the luxury sports and entertainment hospitality company posted $55 million in EBITDA on revenues of +/- $600 million (up from just $23 million in '14).
Howie Long-Short: OLE's UVP is that it can provide exclusive access to events that fans would otherwise have difficulty gaining entrance to. Frank Supovitz (served as the NFL's svp of events from '05-'14) explained that "if a fan is going to spend a lot of money to gain access, then OLE can package the event with a whole bunch of high-margin add-ons and increase the experiential value."
SBJ reported that Live Nation had actually submitted a higher bid for the company, but suggested that the league's relationship with the Endeavor resulted in the P.E. firms accepting the lesser offer; it's being theorized that the current majority owners took less money to maintain "good relations" with the NFL.
RedBird Capital, Bruin Sports Capital and Carlyle Group (CG) bought into OLE for just +/- $70 million back in '15, so they'll realize a +/- 900% return on their investment. As Supovitz noted, "building up a business and then spinning it off is a pretty common occurrence in the private equity world", so it's likely the sale had more to do with cashing in on an investment than an eagerness to get out from under the business, but I do wonder if the triumvirate had reservations about OLE's upside.
Supovitz believes that OLE's ability to move into other sports and entertainment properties is where the "opportunities really lie." That may be true, but acquiring and packaging seats found on the secondary market isn't the lucrative model that the company was founded on (see: Fan's comment below); and neither is giving up a significant portion of the profits to rights holder to gain access to tickets at face value. OLE's margins on Super Bowl packages are "really good" because the company isn't paying a premium to acquire the seats - and they can do the tailgate affordably because they're not paying for space with the event inside the stadium gates - but that's not the case when they're packaging access to other high-profile events; which help to explain why a company with $600+ million in revenue generated just $55 million in profits last year.
If Endeavor is in fact preparing for an IPO, the OLE acquisition may be about "adding valuable earnings" to the company's bottom line, but Supovitz says "that in addition to any economic benefits, the ability to control thousands of tickets to the Super Bowl is powerful - particularly for an agency that represents athletes, coaches and entertainers." In addition to the William Morris Endeavor talent agency, Endeavor counts the UFC, PBR and a video streaming platform (Endeavor Streaming) among its assets.
Fan Marino: OLE has humble beginnings. Back in the 90's, the league would package pro bowl tickets with a gated tailgate party and custom merchandise for fans traveling to Hawaii who wanted an upgraded experience. With the game sold-out, it made for a "great additional revenue stream and it was a very high margin business"; it also allowed the league to re-capture a portion of secondary market ticket sales. By '05 the league realized it could do the same with Super Bowl tickets and began to package seats with hotel rooms - also difficult to find on Super Bowl weekend - and high-end tailgate parties inside the security gates (so fans can party until kickoff). The league sold +/- 1,000 Super Bowl packages the first time around (Super Bowl 40), OLE now sells more than 3,500 annually.
Editor Note: Supovitz has a book - with a focus on crisis management - coming out in May, entitled What To Do When Things Go Wrong.
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Landmark Deal Gives Merchandising/Licensing Distributor +/- 95% of the U.S. College Marketplace
The scale gained via their partnership with Jardine Associates (the largest collegiate merchandising and licensing distributor in the United States) makes Winning Streak - a company that markets, manufacturers and distributes high end premium embroidered flock wool banners and pennants - the top retailer in the category and one of the elite distributors in the $4.6 billion collegiate licensing retail space.
On-Field Success, Scarcity and Novelty Determine if PSL Purchase is a Sound Investment
While fans in Atlanta and New York have come to believe that paying for the right to buy a season ticket is "a scam", the people of Las Vegas have been receptive to the stadium funding mechanism; the Raiders have raised $478.3 million in PSL sales to date, nearly twice their initial $250 million projection.
Is Real-Estate Investment Sports' Next Major Revenue Stream?
Former NFL, NBA, MLB and NHL executive Andy Dolich sees a future where ownership groups are building "condominiums, shopping centers and lifestyle centers - separate from their team's venue (as the A's are considering) - to pad bottom line profits."
Non-Profit Paradox Explains Dramatic Rise in College Coaches Salaries
The dramatic increase in salaries over the last 10-15 years is a byproduct of the real problem facing college athletics; an economic model that systematically encourages acceleration in athletic department spending.
RecruitFocus
DraftKings Reports Mounting Losses, SBTech Takeover Makes Co. 1st 'Vertically Integrated' U.S. Gaming Operator
The DFS turned gaming operator's "need for cash" was confirmed earlier this week when the company filed a detailed financial disclosure with the SEC that reflected $114 million in net losses over the first nine months of 2019.
Endeavor Buys On Location Experiences, Exit Multiple Misreported
While there is no doubt RedBird and Bruin came out ahead on the deal, the 10x return suggested within the SBJ article is laughable (we've heard from insiders that the number is closer to 3x). SBJ failed to account for the company's PrimeSport or Anthony Travel acquisitions among other expenses.
Pro Sports Has Priced Out The Middle Class Fan
The rising cost of fan attendance has put teams - and by proxy pro sports leagues - in a precarious position; while they're maximizing revenues, they're doing so at the expense of seeding new fans.
Unique 'Influencer Engagement Program' Helping Jets "Cultivate Fans"
The Jets 'Influencer Engagement Program' has been a success in terms of facilitating fan development because the content generated by the influencer network reaches a significantly wider audience than the team's fan base. While the franchise regularly speaks to its existing followers through various media channels, "[influencer advocates] enable the club to talk to people who may not necessarily be aware of what it's doing."
Hosting Bowl Game Provides Emotional, Economic Boost for Tucson; Gives Arizona A.D. Chance to "Get in Lock-Step" with Local Movers & Shakers
Of the forty bowl games this season, just six were scheduled to take place on a college campus. But the University of Arizona has found that hosting a bowl game serves as a chance to showcase the University, boosts moral within the community, drives hotel and tourism revenues and helps to raise money for local charities.
Sports Organizations Using Machine Learning Technology to Drive Sponsorship Revenues
Block Six Analytics developed a sponsorship evaluation platform that values sports assets in the same manner "that venture capitalists, private equity firms and investment banks look at investment opportunities."
tmoore87


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2003 vs 1984: Which NBA Draft was Better?
In: NBA
Tagged: LeBron James, Michael Jordan, NBA, NBA Draft
The 1984 NBA Draft has been the standard bearer of my generation. I was 14 years old at the time and I saw the likes of Michael Jordan come into the league that year. It completely reshaped the league in many ways. The style of play certainly changed with the athletic Jordan being able to make moves very few had seen prior. It also was the infant stage of the Chicago Bulls dynasty. Little did we know at the time that the Los Angeles Lakers and Boston Celtics were on the clock.
For a different generation, the 2003 draft had the same type of impact on the league. Some guy straight out of high school named LeBron James came into the league as it's latest savior. Unlike those prior to him, James more than live up to the hype as the league had it's next generational superstar.
Just warning you, this isn't a Jordan vs James debate. That debate has been beaten more than the New York Knicks over the last 20 years.
What we will look at is both drafts in its totality to determine which was the best one.
Top 3 Players in the top 10 selections
If you look at the 1984 Draft, it's easy to pick out the top 3 selections in the top 10. Hakeem Olajuwon (1), Michael Jordan (3) and Charles Barkley (5) all made their impact in the league. Jordan's six rings despite leaving his team in the dust on his baseball excursion, is pretty incredible. It made him the Bill Russell of this day.
Olajuwon would win back to back rings against two centers who made significant impacts in the league. Patrick Ewing would never get that elusive ring as the Rockets beat the Knicks in 7. Shaq O'Neal would later get a ring but the Orlando Magic were outclassed by Olajuwon and the Rockets in series sweep defined by Nick Anderson's two missed free throws.
Barkley is your big numbers guy who never won a ring. His contribution to the league was huge but it is a little bit of a ding that he only appeared in one NBA Finals. Barkley and the Suns would lose to Jordan's Bulls in six games. Other than that, Barkley's playoff teams found more ways to lose a series late in games than win them.
2003's top 3 is a little more challenging but we'll go with James (1), Carmelo Anthony (3), and Dwayne Wade (5). James' eight straight NBA Finals appearances is nothing short of amazing. The number of games, minutes, and pounding shouldn't be ignored. Especially when you consider that in between all the NBA play, he also represented Team USA. That didn't stop him from winning four NBA MVP and three NBA Finals MVP awards.
Wade also has his share of success including winning a ring without James. The 2006 NBA Finals MVP did something most superstars of his ilk wouldn't have done. He took a backseat to James even though, he was in his prime. His sacrifice, along with Chris Bosh, is the prime reason the Heat went to four straight finals where they went 2-2.
Anthony is the Barkley of this draft. Because so many people choose to remember the late stages of Anthony's career, they choose to ignore his time in Denver. Though he doesn't have an NBA Finals appearance, the Nuggets run to the Western Conference Finals after trading Allen Iverson, still sticks out as an incredible achievement. What people can do is punish Anthony for his time with the Knicks. Anthony was stellar despite the incompetence of the front office. It's not all that different than what Barkley went through his last few years in Philadelphia
This was a close that came down to Olajuwon and Wade. Hakeem ended up being the difference.
Category Winner: 1984
Picks 11-20
This one isn't all that close. In 1984, the Utah Jazz selected John Stockton at number 16 and the rest is history. "Stockton to Malone" became a staple throughout the NBA. Stockton would help lead the Jazz to back to back Finals where they lost to the Bulls each time.
In 2003, David West was selected 18th. Outside of that you have names like Reece Gaines and Zarko Cabarkapa.
At least 1984 also has Kevin Willis (11th pick) to go along with Stockton.
Remaining First Round
This gets tricky. The number of teams in the league had increased by 2004 (29 compared to 24) but we're going to roll with it.
In 2004, there were late picks like Boris Diaw (21) and Kendrick Perkins (27). Both won titles and were instrumental in doing so. The Celtics still think they would've won in 2009-2010 had Perkins played in Game 7.
Diaw was instrumental in the San Antonio Spurs championship win over the Miami Heat. The Frenchman would lead the Spurs in assists twice during the series and was credited with the ball movement that game the Heat so many problems.
Picks 20-24 in 1984 are nothing to write home about. Kenny Fields, Tom Sewell, Earl Jones and Michael Young made no impact in the NBA.
Second Round of the Draft
By far, the second rounder with the most accomplished career out of the 1984 draft was Jerome Kersey. the Longwood alum was selected 46th which made him the one before last pick of the round. Kersey would star for the Portland Trail Blazers teams that lost to the Detroit Pistons and Chicago Bulls in the NBA Finals.
Outside of that you have Ron Anderson who had a rather pedestrian 10 year career.
2003 had a few off the bench contributors that made it to the NBA Finals themselves. Kyle Korver and Mo Williams were both taken in the second round that year and found themselves in the NBA Finals with the Cleveland Cavaliers. The second round also produced Zaza Puchulia, Steve Blake, and James Jones.
Rest of the Draft/Free Agency
By 2003, the NBA Draft was down to two rounds as opposed to the 1o rounds it was in 1984. So I will include the rookie free agents from 2003 to even the playing field a bit.
Anyway, there won't be a winner in this category. If you put a gun to my head it would be 2003 solely based on Udonis Haslem.
Category Winner: None
So we end with a 2-2 tie. An appropriate outcome for two drafts that gave so much to the NBA.
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Q: PuTTY closes when opening SMTP over Telnet on certain server I have PuTTY installed on Vista. When I set it to Telnet and put in a host name of SMTP2Go.com and port of 25 the window opens for a second not showing any text then closes. I also tried ports 2525, 587, and 8025, which SMTP2Go says also work with the same result. I use Telnet from the command prompt (which I had to enable from the control panel) to open smtp2go.com with port 25 and it works. I also can use both PuTTY and the other Telnet to connect to outgoing.verizon.net. I am using Frontier for my ISP and it says on their site that port 25 is blocked, and other people have confirmed this, so I find it strange that I can access SMTP2Go on port 25 using Microsoft Telnet. Could SMTP2Go determine what client I'm using and block certain ones?
Also in Microsoft Telnet there aren't line breaks between my commands and the server's response with Verizon, but with PuTTY there are.

A: Just select "Raw" in the "Connection type" field and it will work OK.
From PuTTY's documentation:

A lot of Internet protocols are composed of commands and responses in plain text. For example, SMTP (the protocol used to transfer e-mail), NNTP (the protocol used to transfer Usenet news), and HTTP (the protocol used to serve Web pages) all consist of commands in readable plain text.
Sometimes it can be useful to connect directly to one of these services and speak the protocol 'by hand', by typing protocol commands and watching the responses. On Unix machines, you can do this using the system's telnet command to connect to the right port number. For example, telnet mailserver.example.com 25 might enable you to talk directly to the SMTP service running on a mail server.
Although the Unix telnet program provides this functionality, the protocol being used is not really Telnet. Really there is no actual protocol at all; the bytes sent down the connection are exactly the ones you type, and the bytes shown on the screen are exactly the ones sent by the server. Unix telnet will attempt to detect or guess whether the service it is talking to is a real Telnet service or not; PuTTY prefers to be told for certain.
In order to make a debugging connection to a service of this type, you simply select the fourth protocol name, 'Raw', from the 'Protocol' buttons in the 'Session' configuration panel.

A: That is because there is no SMTP server for "SMTP2Go.com", so Putty is just closing an impossible/non-existent connection. Their SMTP server is actually "mail.treshna.com". Check out this NSLOOKUP for them.
Microsoft Windows [Version 6.1.7601]
Copyright (c) 2009 Microsoft Corporation.  All rights reserved.

C:\>nslookup
Default Server:  google-public-dns-a.google.com
Address:  8.8.8.8

> set q=mx
> SMTP2Go.com
Server:  google-public-dns-a.google.com
Address:  8.8.8.8

Non-authoritative answer:
SMTP2Go.com     MX preference = 5, mail exchanger = mail.treshna.com
>

I was able to use Putty to telnet on port 25 to mail.treshna.com. Also, when I telnet on port 25 to SMTP2Go.com at a command line, as soon as I enter EHLO, it give me this error: "554 SMTP synchronization error" and "Connection to host lost." The server mail.treshna.com does respond to EHLO at a command line telnet on port 25.

redpajama_set_name: RedPajamaStackExchange


text:
Questa North
Peggy Trigg shares love of the natural world through art
Painting how the land feels
The mesas, the mountains, the streams, the cottonwoods and the fields of silver sage are the reasons Peggy Trigg paints. She paints to share this love of the natural world.Trigg combines energies in …
"Dip in the Road" by Peggy Trigg
Posted Monday, December 31, 2018 10:37 am
Painting subjects
I paint a lot on Cebolla Mesa because it's close and I can go on all the different BLM (Bureau of Land Management) roads. I go a lot to Utah and to Arizona, to the reservation land.""
— Artist Peggy Trigg
The mesas, the mountains, the streams, the cottonwoods and the fields of silver sage are the reasons Peggy Trigg paints. She paints to share this love of the natural world.
Trigg combines energies in her life and in her work, which might be considered contradictory, but which she has found to be complimentary: landscapes and abstracts; fine artist and high school teacher.
A rancher's daughter who grew up near Madrid, New Mexico and who now lives with her partner, Mike Ridder in Questa, she said, "I love the energy of the land. I used to ride by myself a lot, all day. I just love the land. I'm trying to capture the energy of the land and the moment."
Trigg credits her dad and her art teachers for encouraging her to follow an artistic path. "I won contests and things like that when I was little." An art teacher helped her to obtain a university scholarship, and Trigg also embraced teaching.
In addition to a career of teaching art at large high schools in Albuquerque where she was grateful for the opportunity to touch the next generation of artists and from which she has recently retired, Trigg has paralleled that success in the gallery world. Currently, her paintings show at La Mesa Gallery on Canyon Road in Santa Fe, Wilder Nightingale Gallery in Taos, the Weyrich Gallery in Albuquerque and the Metier in Dixon.
In 2017 Trigg garnered the Artistic Excellence Award at the Escalante Plein Aire Festival and this year exhibited at the Taos Art Museum at Fechin House, was featured at the Weyrich Gallery, and participated in a juried show.
Plein aire painting has captured Trigg's focus. At least three days a week she feels pulled to paint in the open air.
"I paint a lot on Cebolla Mesa because it's close and I can go on all the different BLM (Bureau of Land Management) roads." Because Trigg also enjoys the open spaces that remind her of growing up on a ranch, she said, "I go a lot to Utah and to Arizona, to the reservation land."
Plein aire was a natural direction for Trigg to explore after retiring. "Being a teacher, I was stuck inside so much of the time," she said. "I wanted to be back there in the elements, feeling the sun on my back, and the wind, and getting bugs in my pictures and dirt."
While this might strike some as extreme, for Trigg it enhances her process. "I really appreciate spontaneity and freshness," she tells. "When you paint in acrylics in plein aire, you have to work really, really fast."
There is, she explains, spontaneity about plein aire painting as long as you don't take it back to the studio and work it to death."
To understand how important the fresh brush stroke is to Trigg, the viewer need only glance at Trigg's studio. Landscapes and abstracts equally fill the space. In the winter when it is difficult to be outside as much, she expands her perspective and loosens her technique through abstract painting.
"When I work with abstracts, I think about meaning," she said. When teaching abstract art, she asked her students, "What does red mean? What does blue mean?"
She introduced students to look at color the way interior designers do when creating a mood. Abstracts, she says, are about feeling and intuition, but also about composition. "You can throw everything down in an intuitive manner, but if the composition's not there, it (the painting) doesn't work."
Trigg not only alternates between her greatest loves in painting, landscapes and abstracts, she melds them. "I try to have a creative approach to landscape. I try to make my pictures look like the land feels, not necessarily how it looks, but how it feels."
"I love the land so much and the abstract." Her landscapes, she feels, "are a marriage between the two."
Trigg's enthusiasm shows itself in her commitment to arts in the Questa area. She and Questa painter Roger Harrington steer the Questa Creative Council which, among many other artistic projects, sponsors the Questa Studio Tour. "It's surprising to me how many artists we have here. There were over 40 on the studio tour. And I really like the traditional arts. That's a really strong aspect of this area," she adds.
Of the art market, Trigg demonstrates a pragmatic optimism. "We have to evolve and change our marketing platforms. You have to work a lot of different markets these days." Trigg does well on many. She sells well on her website and on the studio tour and performs demonstrations at the gallery in Santa Fe.
Many more images and information is available at peggytrigg.com.
peggy trigg,	artist,	painter,	fine art,	questa north column,	anna racicot,	the taos news,	taosnews,	tempo magazine


text:
without photo
Daily Sabah >
Columnists >
Kılıç Buğra Kanat
Rising intolerance and the future of European politics
KILIÇ BUĞRA KANAT @KilicKanat
Published 26.03.2017 23:11
As Turkuvaz, we are processing your personal data, and at the same time, using our best efforts to protect your data by taking the technical and administrative measures prescribed by laws. You may find the informative text in respect of your personal data processed, by visiting the data policy page.
COLUMNS Forgetting the suffering of Syrian refugees
COLUMNS Threat of far-right parties in Europe
COLUMNS European tolerance for the 'other' crumbles
Since the beginning of the rise of far-right political parties in Europe, I have written in this column that the threat posed by these parties is due to things beyond the risk of their electoral victories. Although it has been repeated in multiple instances, it is necessary to re-emphasize the real risk of the political discourse of these parties since almost every week we experience a new symptom of this discourse on minorities and different voices in Europe. The acceleration of this phase in far-right political discourse in Europe may not be related solely to the elections in various European countries this year, it may be a long term problem and continue to have far reaching implications for society and politics in Europe.
The risk of these parties and the rhetoric they introduce in European politics has more to do with the possibility of the evolution of political discourse in these countries in a way that allows more space for intolerance, and in some extreme forms, xenophobia. A dangerous part of the rise of these political parties is the use of parallel discourses in different countries and the establishment of transnational anti-immigrant and anti-Muslim rhetoric. Opposition to the Turkish accession to the EU, which has been the mantra of far-right political parties, has recently been complemented by anti-Syrian immigration attitudes and increasing religious discrimination.
The mainstreaming of this rhetoric generates major risk beyond simply cultural diversity and the European integration process. Minorities living in European countries feel threatened by the mobilization of certain segments of society and worry that that rhetoric may not end following the elections. The demobilization of now increasingly empowered and emboldened far-right rhetoric may not be easy. Once these feelings are indoctrinated and planted in a society, it can take a long time to demobilize these feelings. Thus, what happens in the election campaigns of these parties does not stay in the election campaigns but impacts the society as a whole long after campaign season.
In recent weeks, due to the crisis between Turkey and the Netherlands and attempts by several European countries to prevent some Turkish politicians from addressing Turkish citizens about the upcoming referendum, the implications of the rise of this political rhetoric have begun to manifest. The spread of these ideas has started to influence the overall political atmosphere and rhetoric in these countries, as well as spread to institutions in Europe.
Last week, the European Parliament made the controversial decision to ban the distribution of Daily Sabah in the institution. It is still not clear how an institution can make such a scandalous decision; however, it is very concerning once this decision is analyzed in the context of rising intolerance in Europe. Since January, many European analysts have argued that 2017 could be a critical juncture for Europe due to important elections in certain European countries, especially after the Brexit referendum. A major part of this juncture will have something to do with the reaction of mainstream European parties and politicians to this intolerant discourse. It will probably be one of the most important tests for Europeans.
Disclaimer: All rights of the published column/article are reserved by Turkuvaz Media Group. The entire column/article cannot be used without special permission even if the source is shown.
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İslamophobia


text:
PUP's Marconi Leal rushed to KHMH due to COVID-19
Robbery on the rise in South, businessman targeted in Dangriga
City man shot in Seine Bight
Posted: Tuesday, September 7, 2021. 3:26 pm CST.
By Aaron Humes: Gunfire was reported overnight in a quiet corner of the country – the Garifuna village of Seine Bight, Stann Creek District.
According to police, they found Akeem Augustine, 26-year-old Belizean unemployed of a Belize City address who was seen suffering from what appeared to be a gunshot wound to the body at the village polyclinic just before 9 p.m. Monday night.
Initial investigation revealed that at about 8:00 p.m., Akeem and a female were walking in a dark area heading to their apartment in Seine Bight Village when they were approached by an unknown man who fired several shots at him. Augustine began to run but was followed by the culprit who continued to fire shots at him, which resulted in his injury.
Jerome Crawford, accused of murdering his boss makes second court appearance; Body of victim still not recovered
International News: Over 50 unaccompanied Guatemalan children found in migrant truck in Mexico
San Ignacio and Santa Elena House of Culture calls Cultural Engagement Community Meeting for February 7
Rheema Emmanuel wins 'Belize is Antimicrobial Aware!' Video/Poster Competition for tertiary-level students
Belize supports Taiwan's Participation at the 76th World Health Assembly
Belize Weather: Last of cold front crossing today
Please email us your news at news[email protected]


text:
Q: Unbinding a listener/handler from an Element in jQuery I am using a jsonSuggest jQuery plugin to show autocomplete data in an text box on the website.
To turn it ON I call:
$('input#editbox').jsonSuggest({url: someurl, onSelect:callbackFunc});

However I am unable to deactivate the auto-suggest once the it's been turned on. In particular, when I make another call for the same editbox with different parameters, 2 autocomplete boxes appear. So I would like to turn it OFF before turning it ON again with new parameters.
The code is only about 300 lines and available here
https://github.com/cootetom/jQuery-JSON-Suggest-Search-Box/blob/master/jquery.jsonSuggest-2.js
I ran through it with a debugger, but can not find the point where a listener is attached or a "bind" is made. Admittedly I am not 100% profficient in jQuery.
Thanks in advance
PS: Could this be part of the solution?
Calling
$('input#editbox').off("keyup");
$('input#editbox').off("keydown");

before calling
$('input#editbox').jsonSuggest(...)

again?

A: https://github.com/cootetom/jQuery-JSON-Suggest-Search-Box/blob/master/jquery.jsonSuggest-2.js#L317
Thats where all the binding happens. So you need to unbind keyup, keydown, blur and focus events using .off like $('input#editbox').off('keyup');


text:
package grammar.model.verbs;

import java.lang.reflect.InvocationTargetException;
import java.lang.reflect.Method;
import java.util.List;
import java.util.regex.Matcher;
import java.util.regex.Pattern;

public class CliticOrder {
	private final String name;
	private final If predicate;
	private final List<Condition> conditions;

	private CliticOrder(String name, If predicate, List<Condition> conditions) {
		this.name = name;
		this.predicate = predicate;
		this.conditions = conditions;
	}

	public boolean matches(Conjugation conjugation)
			throws IllegalArgumentException, NoSuchMethodException, IllegalAccessException, InvocationTargetException {
		return predicate.eval(conjugation);
	}

	public String inflect(Conjugation conjugation) {
		for (Condition condition : conditions) {

		}

		return null; // FIXME
	}

	public String getName() {
		return name;
	}

	public static class If {
		private final boolean conditional;
		private final String conditionStr;

		private If() {
			conditional = false;
			this.conditionStr = null;
		}

		private If(String conditionStr) {
			conditional = true;
			this.conditionStr = conditionStr.trim().replaceAll("\\s+", " ");
		}

		public boolean eval(Conjugation conjugation)
				throws NoSuchMethodException, IllegalAccessException, IllegalArgumentException, InvocationTargetException {
			if (!conditional)
				return true;

			String[] ors = conditionStr.split(" or ");
			if (ors.length > 1) {
				boolean rtn = false;
				for (String or : ors) {
					rtn |= new If(or).eval(conjugation);
				}
				return rtn;
			}
			String[] ands = conditionStr.split(" and ");
			if (ands.length > 1) {
				boolean rtn = false;
				for (String and : ands) {
					rtn &= new If(and).eval(conjugation);
				}
				return rtn;
			}

			Pattern p = Pattern.compile("^([a-zA-Z]+)\\(\\)$");
			Matcher m = p.matcher(conditionStr);
			if (m.matches()) {
				String methodName = m.group(1);
				Method method = conjugation.getClass().getMethod(methodName);
				return method.invoke(conjugation).equals(Boolean.TRUE);
			}
			p = Pattern.compile("^([a-zA-Z]+)\\(\\) (==|!=) ([a-zA-Z]+)$");
			m = p.matcher(conditionStr);
			if (m.matches()) {
				String methodName = m.group(1);
				Method method = conjugation.getClass().getMethod(methodName);
				String result = (String) method.invoke(conjugation);

				String operation = m.group(2);
				String str = m.group(3);

				if (operation.equals("=="))
					return result.equals(str);
				else if (operation.equals("!="))
					return !result.equals(str);
				else
					throw new IllegalArgumentException("Invalid operation in clitic order condition: "+operation);
			}

			throw new IllegalArgumentException("Could not parse condition: "+conditionStr);
		}
	}

	public static class Then {
		private String thenClause;

		public String eval(Conjugation conjugation) {
			String str = thenClause
				.replace("{prefix}", conjugation.getPrefix())
				.replace("{auxiliary}", conjugation.getConjugatedAuxiliaryStr())
				.replace("{verb}", conjugation.getConjugatedVerbStr());

			return str; // FIXME
		}
	}

	public static class Condition {
		private final If ifObj;
		private final Then then;

		private Condition(If ifObj, Then then) {
			this.ifObj = ifObj;
			this.then = then;
		}

		private Condition(If ifObj) {
			this.ifObj = ifObj;
			this.then = null;
		}

		public If getIf() {
			return ifObj;
		}

		public Then getThen() {
			return then;
		}
	}
}

redpajama_set_name: RedPajamaGithub


Honestly, I didn't know what to expect from this book, but I couldn't put it down. Haddon masterfully captures the behaviors of a person with Asperger's. Christopher thrives on logic and order, he's brilliant with math and solving puzzles. He's emotionally detached from people, he doesn't understand the subtleties of emotions and body language. He doesn't like talking to most people because they do and say things that he genuinely doesn't understand, which is frightening to him. He loves the idea of being an astronaut living alone in outer-space, he finds absolute safety in solitude. We learn all of this as Christopher narrates the story of his detective work. It's fantastic how quickly Haddan inspires empathy for Christopher. Behaviors exhibited by people with an Autism Spectrum disorder are often seen by people as extremely odd or even disruptive. They can't look you in the eye, they cover their ears and rock back and forward. They scream in public for no apparent reason. Through Christopher we better understand the whys of his behaviors, he's living in a world in which he simply doesn't always fit.
The Curious Incident of the Dog in the Night-Time isn't just about a murdered dog, it's really so much bigger. The story is so compelling because Christopher pushes himself so far out of his comfort-zone, he does things that terrify him. Ultimately, he discovers far more than who murdered Wellington.
Curiously enough, the book club at my work selected this novel a few months ago. I didn't pay it much mind because… well, I don't think my tastes are very similar to my co-workers. *smirk* However, after reading your summary, I'd be more inclined to read the book.
Thanks! I've been considering this book for a while. I'll give it a go.
This seeing-through-the-eyes-of-another reminds me of the movie 'Strange Days'. With the neural interface, experiencing live recordings of other people. Since I was young I wondered what colors other people saw that they learned to call 'red', or 'pink', or 'orange', versus what I personally visually perceived. It fascinated me, the way our perceptions can be so different with the same stimuli. Which is why I ended up studying Neuroscience.
I know of many experiments carried out years ago that utilized a number of neural electrical detectors to allow people with paralysis control various devices just by thinking certain ways/things. I'm wondering how much more efficient this might be for you instead of using your switch. Of course, I have no idea of cost or availability. Just an FYI for you. =) Best wishes.
I just finished reading this book yesterday, and I have to say that I very much enjoyed it. I've seen it in the bookstores for years, but never picked it up because the blurb on the back really doesn't give the story justice. Over the weekend I was at a used bookstore and saw it on a shelf for $1. I remembered your review and decide to give it a shot. I'm glad that I did. It was a quick read and much better than I expected it to be.
Hey thanks, I've got a nephew with Asperger's. I had a dream about him the other night. So, now I'll read the book. And maybe I'll have another dream. And maybe he'll read the book. And maybe he'll have a dream. And maybe we'll meet in dreamland and it will be easy and happy, but not shallow, no, as deep as the sky… as deep as the sky.


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Anyway, to the point...this video is from the feature shoot for latest issue of Oxygen, one of the leading women's fitness magazines. Unfortunately every online source failed to credit or link to the photographer responsible. Not this guy. Robert Reiff is the man behind the camera. The thing I want you to pay attention to is the use of light for contouring. Those of us in the fitness industry do this all the time. I'm not sure how prevalent it is in other genres, but when defining a body it is something you have to master. In the case of these images the rim light is kept soft and feathered gently. This particular publication prefers to keep their images feminine and approachable which is why that kind of light was chosen.
The harder the light, the more intense the definition. You're going to have to pause the video a few times to get a solid look at what is going on but, if you want to work with physiques...you need to know this.
Can someone tell me what the key light is?
The Key is a Fresnel Spot, not sure if its profoto or not, it's too big to be the Hard Box as that actually sits on the head while the Fresnel has a yolk that mounts to the stand.
Can someone tell me what the fill light is?
My best guess is they're using Profoto... The main looks like the Hardbox and the others look like B1 monos.
Great post, I love seeing BTS of other's shoots...thanks! It's also a great way to bring more attention to yourself as a photographer, never know when someone is going to see it and say, "hey, remember that guy that shot that fitness model? We need something like that."
Oh I didn't even know Oxygen was back! It went under in 2013 and I bought the "last" issue.
Yeah, they sold to Active Interest Media shortly after the owner died. Robert was an awesome guy, I'm really glad they were able to keep his publications going.
Lost one minute of my life haha.


Gerður Kristný was born in 1970 and brought up in Reykjavík. She won the Icelandic Literature Awards in 2010 for her book of poetry Blóðhófnir (Bloodhoof) which is based on an ancient Nordic Myth, told in the Eddic poem Skírnismál about the attempt of the Nordic fertility god Freyr to fetch the poet's namesake Gerður Gymisdóttir from her far away home as his bride. Gerður Kristný has published collections of poetry and short stories, novels, books for children and a biography for which she recieved the Icelandic Journalism Award in 2005. Other awards for her work include the Icelandic Children's Choice Award in 2003, The Halldór Laxness Literary Award in 2004 and The West-Nordic Children's Literature Award in 2010. Gerður Kristný lives in Reykjavík but travels regularly around the world to present her work, giving readings in places like Kampala, Java, New York, Maastricht and Colgata.


HMI Is Again Anticipating Putting On "A REALLY BIG – AND DEEP – SHOW" at The World Of Concrete in Las Vegas.
There are exactly 62 days until the curtain goes up for "Greatest Show in Concrete." Will I be seeing you in Las Vegas? 2019 will represent the 40 consecutive year HMI has been participating in the BIG SHOW!
The greatest show deserves the greatest demonstrations. Again in 2019 HMI is planning new and exciting exhibits and presentations. This year we will feature our DEEP INJECTIONS SYSTEM & PRODUCTS. You may think you are at the circus as we climb to heights to inject our patented hydrophobic foam deep through our patented injection pipes utilizing the ELITE ONE gun. Demonstrations will be continuous over three and half days from January 22nd through January 25th. We plan to break the record this year with over 100 flawless demos and lifts.
HMI will again be in the SILVER LOT at booth 041045. Nearly the entire HMI team will be at the show. Plan on stopping by to visit: Jeff, Brian, Jen, Jason, Kip, Teddy, Jesse, Paul and ME! The team is excited. For us the World of Concrete is like a reunion as we see old friends from Discovery & Training Seminars over the past decade. Catching up with members of the HMI family on; business, life and home clans is what is often most important about attendance.
HMI has coupon codes available for show registration discounts. Visit our WEB SITE or call us and save.


text:
Q: Dep Ensure Not Working Following New Computer Install From Git I just finished up my first big project for a software engineering company, but when I tried to install the code on my personal computer tonight I am having trouble with Go. I set up my GOPATH and GOROOT and things seem to be okay. Here are those:

Everything worked GREAT today on the work computer, but now its just a pain. I'm getting all kinds of errors, but the biggest is that it "cannot find package." It is also giving me an error for "use of internal package not allowed" when it worked just fine on my work computer. I worked primarily in the front end so I'm a little ignorant on the subject, but I texted a co-intern and he didn't know either. I am thinking its my Go installation.
We used dep ensure for the dependencies and it is looking in the wrong place. for the vendor files. Here is an example of the file structure:

I would LOVE some help with this because I was so excited to show this thing off we worked so hard on, If anyone is interested here is the GitHub link!
https://github.com/acstech/doppler-events
Thank you everyone!