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How to help a foster child with their anxiety
By Linda Hill
In Advice for Carers
How to help a foster child with their anxiety2020-09-302021-01-04https://likafamilyfostering.co.uk/wp-content/uploads/2018/06/lika-family-fostering.pngLiKa Family Fosteringhttps://likafamilyfostering.co.uk/wp-content/uploads/2020/09/anxiety-in-foster-children.jpg200px200px
Anxiety in a foster child can be crippling. Not just for the young person experiencing it, but for you, as their carer, not knowing how to help.
Dealing with anxiety in children and, specifically, giving young people mechanisms to cope is the idea behind Cheeky Worries — a special book written by Dr Anna Smith, a clinical psychologist, and Dr Patrick Davey, a consultant psychiatrist.
Both work in the NHS and specialise in treating anxiety disorders. Dr Patrick has also worked before with young people fostered with LiKa carers.
So, we thought we'd ask this pair of experts for their best advice on how foster carers can help young people handle their anxiety.
What sorts of anxieties or anxiety-related issues do we tend to see in young people? Do we know where they come from?
Dr Anna: What you tend to see is younger children have more concrete phobias. So, fear of dogs or a fear of monsters.
As they get older, they might start fearing things that are less concrete. They might start developing separation anxiety, where they feel anxious about being apart from their main caregiver, for example. They might worry about people dying.
As you get into early teens, you're more often seeing worries like Obsessive Compulsive Disorder. So, that's an anxiety about contamination or dirt or germs.
Then, as you get into mid-teens, it's more things like social anxiety, anxiety about body image, and that kind of thing.
How do you help a foster child experiencing anxiety?
Children in foster care have usually experienced some kind of trauma. How might that create anxiety and how can we identify it?
Dr Anna: For people who've experienced trauma, it's about helping them link and understand how those experiences might have impacted on the way they interact with the world now.
For example, if they have experienced a traumatic loss, they might be more anxious about things like separation, the possibility of having another loss in their life, or the possibility of being left again in some way. So, their fears, later in life, might mirror some of their earlier experiences, if that makes sense?
The thing is, there's no single cause of that anxiety. Five children can go through exactly the same event and all respond in different ways.
But I suppose what we pick up on are signs in the child of something different, like in their behaviour, for example. A child who was really happy to go to bed alone might suddenly start to appear a bit more needy at night-time. They might keep coming downstairs for something, when they should be asleep in bed. It's not necessarily that they would ever say 'I feel anxious'.
What the foster carer needs to be doing is tuning in to subtle changes in behaviour that might indicate there's something that is making the child feel anxious and behave in a different way.
READ MORE: 4 top tips to help foster carers manage their behavioural triggers
What sorts of behavioural changes or signs are we talking about?
Dr Patrick: We talk a lot in the book about people not doing the things they might normally want to do.
So, for example, not wanting to go to school in a child who'd normally been quite happy going to school, not wanting to meet their friends in a child who had previously enjoyed seeing their friends, not wanting their foster carer to go out when before they might have been quite happy for their carer to go out.
Also, look out for any physical health complaints — a grumbling stomach, aches and pains — which suddenly appear around a certain period of time, or ahead of something. Recurrent stomach ache, that kind of thing.
The difficulty is children are not always able to explicitly say 'this is my anxiety', but saying they have a funny sensation in their body repeatedly, or complaining of a tummy ache, is much easier.
It's important to find a way to open a dialogue with your foster child.
Image Courtesy of: Cheeky Worries
If you observe this kind of behaviour in your foster child and you think it might be anxiety, what should a foster carer do next?
Dr Anna: You want to be able to find a way to open a dialogue, because what we want is for these experiences to be completely normalised.
You might say: 'It's totally fine and normal that you'd feel anxiety about this. I understand what anxiety is; I have anxiety myself. Let's talk about anxiety; let's not keep it something that's really hidden away'.
The first thing you want to do is just gently explore and to ask things like, 'Oh, you used to love going to the park and it seems like you're not so keen to go to the park anymore. Is there anything different about the park, or is there anything you're worried about going to the park'?
We always say don't be scared to ask the questions. Ask if something is making them feel scared or worried. It's actually just so important to just dive in.
Children don't always want to tell the truth in these situations, do they? How do you know if they're maybe not telling you what they're actually feeling?
Dr Anna: You can use a technique where you name an anxiety of your own, to model that it's okay to talk about these things.
Or you could say 'some children might feel a bit worried if… [insert what you think the worry might be here]. Do you ever have worries like that'?
Often, we don't want to do that because we worry we're going to be planting things. But we're really trying to validate these experiences for the children.
If a caregiver can encourage a foster child to share their anxiety, then they can acknowledge and validate it.
Once you've opened that door, what techniques can carers use to help a young person deal with their anxiety?
Dr Anna: The important thing in younger children is just getting them to share their anxiety. Then the caregiver should acknowledge it, give them a hug, validate it.
We're not trying to reassure them by saying, 'Oh no, don't worry about that, nothing is going to happen'. Reassurance doesn't actually work. It keeps the anxiety going. But it's also not true — something bad could happen. You have to allow that anxiety to be there, to be okay, and to be okay to talk about it. Younger children are quite resilient.
Acknowledge that it's really sad, it's really scary, or it must make them feel horrible.
With older children, you're trying to get them to build their own resilience and build their own little set of coping strategies. So, it's about acknowledging, 'OK, so you feel really anxious and horrible. What can we do right now that might help you feel a little bit better'?
Then offer suggestions. 'Would that be sitting down and doing a game with me? Would that be doing some baking? Should we go for a walk'?
You're not trying to say 'I'm going to take this anxiety away', which is what lots of adults want to do, but you're saying, 'it's all right; you can cope with this anxiety; you can manage it. It feels horrible. It feels scary, but let's try and do some things to make our bodies feel better in the moment'.
READ MORE: Foster carers: Understanding the teenage brain
What does success look like? How do you know you've made a change for the better in that moment?
Dr Anna: The sad fact is you can't get rid of anxiety. But the change would be that a child is able to still be as involved in life as they would like to be despite their anxiety.
Acknowledging sometimes that they have anxious thoughts, and being able to say to their caregiver, 'I feel a bit nervous about going to this party', is success. It means you can have a discussion about it, but that they still go on to the party. So that even if the anxiety's there, which it might be for a lot of children who are in a position where they're being fostered, the important thing is that I can still carry on with their life.
You can't get rid of anxiety, but success means a child will be able to still be as involved in life as they would like to be.
When the child comes back from the party, to use your example, do you have a follow-up conversation about how it went?
Dr Anna: One hundred percent, yes. Because for the child, we want them to learn it's okay to say 'I felt a bit nervous, but then I talked to my friend and actually I felt a bit better, and then we played a game and that was quite fun'.
Then it's up to the foster carer to say 'Brilliant. You did so well there because you felt anxious and you didn't feel like going, but you did it anyway. You were able to carry on doing a thing you wanted to do, even though there was this whirl of anxiety there at the same time'!
READ MORE: How to transfer from your current agency to LiKa
If a foster carer is trying these techniques but doesn't feel like they're making progress, what should they do?
Dr Patrick: It's important to remember that you've got all these skills. It's about human-to-human communication, letting the young person know you're there, that they can talk to you about it, that you are curious and want to understand what they are finding difficult.
In terms of when to get help, I would suggest that if things are persisting and continuing to have an impact on a child's ability to enjoy their life fully then it's worth considering getting some advice. There are some very good online resources or you can speak to your GP for their thoughts and signposting.
Helping your foster child with their anxiety is about persisting.
Do you have a last piece of advice for foster carers helping their young person deal with their anxieties?
Dr Patrick: Just that it's normal to have anxiety. Everyone experiences it at different times — adults and kids — and we have to be careful we don't pathologise (treating as abnormal) normal, perfectly healthy worry and anxiety.
Dr Anna: The other thing is, in this particular group of young people, the anxiety might present in all sorts of different ways. Particularly if you've had quite a difficult early life, or multiple caregivers at an early age, your anxiety might show itself in slightly different ways.
It can be quite easy to just view it as a behavioural problem, but it's always worth asking yourself 'is this driven by anxiety? What is this young person thinking or feeling that might be making them act in this manner'?
You can find out more about Cheeky Worries, here.
LiKa foster carers have 24/7 support to help them with any issues their young person might be experiencing, including anxiety.
If you're in south or east London and you're interested in becoming a foster carer, give the helpful team at LiKa a call on 020 8667 2111. We're here to answer all your questions.
We're in the London boroughs of Croydon, Sutton, Bromley, Merton, Lambeth, Westminster, Wandsworth, Lewisham, Southwark, Islington, Camden, Hackney, Tower Hamlets, Ilford, City of London, Haringey, Newham, Redbridge, Barking and Dagenham, Hammersmith and Fulham, Kensington and Chelsea.
Anxiety, Cheeky Worries, Dr Anna Smith, Dr Patrick Davey
Daily Logs – Everything a foster carer needs to know
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What is a systemic approach to foster care? May 24, 2019
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LOS ANGELES, CALIFORNIA - JANUARY 18: Caitlyn Jenner speaks at the 4th annual Women's March LA: Women Rising at Pershing Square on January 18, 2020 in Los Angeles, California. (Photo by Chelsea Guglielmino/Getty Images)
Caitlyn Jenner Laments Homeless Tents In Beverly Hills
by Dongyoon ShinJune 8, 2021, 6:07 pmJune 8, 2021, 6:07 pmJune 8, 2021, 6:07 pmDaily Digest, News, News Feed >
California Republican gubernatorial candidate Caitlyn Jenner said Friday that the tents in Beverly Hills are the latest sign of the deterioration of the state.
"When you drive in Beverly Hills and you look at the park, and there's tents in the park, we have to look at that issue very seriously," Jenner told Fox LA on Friday. "But to be honest with you on a big picture, living in a tent in a park in Beverly Hills, it's just different today. Before, the homeless were all downtown. I'm sure you've been downtown. I mean, you walk down there, and it's just like so tough, so difficult on these people," she added.
Gov. Gavin Newsom (D-California) is facing a recall election this year, and 43 people have declared their candidacy. However, only a few have recognizable political experience.
Jenner has no experience in running for office. And despite her celebrity as a former Olympic gold medalist and a Kardashian family member, she is polling at a mere six percent, according to the Berkeley Institute of Governmental Studies survey in May.
During the interview, Jenner also claimed that 50 percent of all homeless people in the U.S. live in California. A count from a federal report last year estimated the number was closer to 28 percent.
Caitlyn Jenner Gavin Newsom Recall Gavin
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text:
Gut Pathogens
The O-antigen negative ∆wbaV mutant of Salmonella enterica serovar Enteritidis shows adaptive resistance to antimicrobial peptides and elicits colitis in streptomycin pretreated mouse model
Sangeeta Jaiswal1,
Niladri Bhusan Pati1,
Manupriyam Dubey1,
Chandrashekhar Padhi1,
Prakash Kumar Sahoo1,
Shilpa Ray1,
Aryashree Arunima1,
Nirmal Kumar Mohakud2 &
Mrutyunjay Suar1
Gut Pathogens volume 7, Article number: 24 (2015) Cite this article
Salmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice.
In the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization.
Deletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, ΔwbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt. In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant.
OAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. ∆wbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model.
Salmonella are Gram-negative, intracellular bacteria that cause diseases ranging from acute gastroenteritis to typhoid fever, posing a significant threat to public health globally. Infections with non typhoidal serovars of Salmonella enterica, predominantly S. enterica serovar Enteritidis (S. Enteritidis) and S. enterica serovar Typhimurium (S. Typhimurium) are more frequent and occur in both developing and industrialized nations [1]. Salmonella employs a number of virulence factors to successfully colonize and replicate inside the host. The most critical virulence determinants of Salmonella infections are Salmonella pathogenicity islands (SPI), of which SPI-1 and SPI-2 play crucial role in invasion and intracellular replication respectively. Apart from these, numerous additional virulence factors such as pilli or fimbria [2], flagella [3], lipopolysaccharide (LPS) [4] etc. are required for a successful infection. Among these factors, role of LPS in Salmonella virulence has been established by several studies [5–7]. LPS is a major structural component of the outer membrane of all the Gram-negative bacteria and plays a critical role in the bacterial pathogenesis. It consists of three distinct domains: lipid A, core oligosaccharide (OS) and O-antigen polysaccharide (OAg). The different components of LPS interact with different parts of host cells and contribute to bacterial pathogenesis. For example, different sugars of outer core interact with epithelial cells, whereas, lipid A interacts with the TLR4, a surface receptor of immune cells. While lipid A and core OS structures are fairly conserved, the OAg is highly variable, leading to serological specificity among Gram-negative bacteria. OAg is a modular assembly of oligosaccharide units that varies with respect to the sugar composition and number of their modal repeats [8]. Typically, in all the Gram-negative bacteria, this region comprises of 16 to more than 100 repeats of oligosaccharide units containing 4–6 monosaccharides each. Bacteria lacking OAg are called rough. The repeating unit of OAg of Salmonella mainly consists of three hexose sugars namely mannose, rhamnose, galactose and one dideoxy hexose as the fourth component [9]. Mannose, rhamnose and galactose form the backbone of the OAg and are conserved across different serovars of Salmonella. The dideoxy sugar linked (α-1, 3) to mannose residue varies among different serovars. Based on the agglutination by antibodies against different O-antigens, Salmonella have been grouped into six serogroups namely A, B, C1, C2, D and E. The group A (e.g. S. Typhi) contain paratose (3,6-dideoxy-d-ribo-hexose), group B (S. Typhimurium) has abequose, whereas group D (e.g. S. Enteritidis) contain tyvelose (3,6-dideoxy-d-arabino-hexose) as fourth component of OAg repeating unit [10]. A schematic representation of the biosynthetic pathway of these sugars is shown in Fig. 1a. In S. Enteritidis, enzyme paratose synthase (Prt, formerly known as RfbS), synthesizes paratose, which is further epimerized by the enzyme CDP-tyvelose-2-epimerase (Tyv, formerly known as RfbE), to tyvelose which is then transferred to OAg repeating unit by tyvelosyl transferase (WbaV, formerly known as rfbV) (Fig. 1a). The genes responsible for OAg biosynthesis are generally found on the chromosome as an OAg gene cluster and genetic variation in this cluster reflects the structural variations of OAg across Gram-negative bacteria. This gene cluster encodes proteins that can be further categorized into three groups [11]. First group involve proteins that synthesize nucleotide sugar precursors. Proteins of the second group are mainly glycosyl transferases (GTase) that build sequentially, the OAg-repeating unit on the carrier lipid, undecaprenyl phosphate (UndP). The third group of enzyme is mainly OAg processing enzyme involved in the polymerization and translocation of OAg across the membrane. The enzymes, Tyv and Prt are involved in biosynthesis of CDP-sugars whereas WbaV is a GTase. The location of the above genes on chromosome has been depicted in Fig. 1b.
Schematic presentation of final steps of dideoxy sugar biosynthesis, location of genes on chromosome and LPS profile of wild-type S. Enteritidis and its isogenic mutants. a CDP-paratose is synthesized by CDP-4-keto-3,6-dideoxy-d-glucose by the action of prt which is epimerized by Tyv to tyvelose. After synthesis, tyvelose is transferred to OAg backbone of mannose (Man), rhamnose (Rha) and galactose (Gal) by WbaV. CDP-abequose is also synthesized from same CDP sugar intermediate as CDP-paratose. b Location of genes on chromosome. The regions enclosed by the black bar were deleted with the help of lambda red recombinase system. White arrow indicate that genes are located on negative strand. c LPS from wild-type Salmonella as well as the mutants were isolated using the protocol described in materials and methods and separated on polyacrylamide gel electrophoresis (PAGE) gel using tricine-SDS buffer system. LPS were visualized by silver staining. c∆ indicates the mutants are complemented with the corresponding genes
Deletion of the genes involved in nucleotide diphosphate (NDP) sugar biosynthesis renders bacteria unable to synthesize complete OAg [12]. For example, deletion of galE or pmi genes involved in NDP sugar biosynthesis, results in the truncation of LPS which can be further restored via trans-complementation [12–14]. Since, both prt and tyv are involved in CDP-sugar biosynthesis [15, 16], their deletion could possibly affect OAg biosynthesis. OAg biosynthesis and assembly is a highly co-ordinated process and is accomplished step by step. Interference at any step may not only affect the OAg biosynthesis but also other cellular processes as sugar intermediates are synthesized from precursors obtained from common metabolic pathways [16]. In the present study, we have blocked tyvelose biosynthesis and its transfer to OAg repeating units by gene deletion (tyv, prt and wbaV) and investigated their effect on S. Enteritidis virulence. Interestingly, we found that deletion of tyvelosyl transferase gene (wbaV) resulted in increased resistance against antimicrobial peptides (AMPs) and decreased degree of attenuation in the rough strain of S. Enteritidis as compared to other OAg-negative strains.
Deletion of genes involved in OAg biosynthesis from the genome of S. Enteritidis results in the production of OAg-negative LPS
In order to investigate the effect of deletion of genes involved in biosynthesis of OAg, the ΔwbaV, Δtyv, and Δprt gene were deleted from the genome of S. Enteritidis and LPS from the respective deletion mutant was analyzed by SDS PAGE and silver staining. It was observed that the strains were unable to synthesize OAg repeating units (Fig. 1b). However, the synthesis of lipid A and core region remained unaffected. To prove that the deletion of genes had no polar effect on downstream genes, mutant strains were complemented with their respective genes with the help of plasmid listed in Table 1. Upon complementation, all the strains synthesized complete OAg repeating unit (Fig. 1c).
Table 1 Strains and plasmids used in this study
OAg-negative mutants showed significant impairment in the motility
Bacterial strains lacking OAg are known to be less motile as compared to those with long OAg repeating units. To confirm if the OAg-negative mutants used in this study were also less motile, the motilities of the mutants were analyzed by soft agar assay. All the OAg-negative mutants were found to be less motile as compared to the wild-type and showed almost two fold reduction in their motility (Fig. 2a).
Assessment of motility and sensitivity to antimicrobial peptides and serum. a Wild-type Salmonella and its isogenic mutants were placed at the center of the agar plate containing 0.3 % agar and incubated for 8 h. Diameter of growth zone was measured in centimeter (cm). Experiments were performed in triplicates at three independent occasions. b For AMP sensitivity assay, approximately 1 × 107 CFU/ml bacteria in PBS were incubated with or without antimicrobial peptides (1 µg/ml polymyxin B, 5 µg/ml protamine, 10 µg/ml LL37, and 2 µg/ml cecropin) for 1 h at 37 °C. The number of surviving bacteria was determined by plating serial dilutions on LB agar plates and survival percentage was calculated. Statistical analysis was performed using two-way ANOVA. c For analysis of serum resistance 1 × 107 CFU/ml of bacteria was incubated with or without 50 % of normal human serum and serial dilutions were plated to enumerate the number of surviving bacteria. Statistical analysis was performed using student t-test. The survival of Δprt and Δtyv mutants was compared with that of ΔwbaV. Level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant. Error bar indicate the standard deviation of three independent experiments
OAg-negative mutants showed differential sensitivity to AMPs and complement mediated killing
The structure and composition of LPS is known to affect the susceptibility of bacterial strains to various cationic AMPs [17, 18]. Therefore, we evaluated the sensitivity of these mutants to different cationic AMPs. The survival efficiency of the mutants was compared to that of the wild-type (normalized to 100 percent). All the mutants showed increased susceptibility to AMPs tested in this study as compared to the wild-type. Interestingly, the ΔwbaV mutant showed significantly increased resistance to all the AMPs as compared to other OAg-negative mutants (P < 0.001), which was reduced up to the level of wild-type upon complementation (Fig. 2b).
To further characterize the mutants, the sensitivities of mutants to complement mediated killing was determined. All the OAg-negative mutants were found to be more susceptible to killing by serum as compared to wild-type strain. The OAg-negative strains showed 50 % or less survival in serum when compared to the wild-type (normalized to 100 %). The ΔwbaV mutant, however, showed significantly increased survival (P < 0.05–0.01) as compared to other two OAg-negative mutants (Δprt and Δtyv) (Fig. 2c).
The ΔwbaV mutant showed increased adhesion and invasion efficiencies
To study the effect of deletion of above genes in adhesion and invasion, the ability of different OAg mutants to adhere and invade HCT-116 cells was compared. OAg- negative mutants attached and invaded HCT-116 cells more efficiently as compared to the parental strain. Among all the three mutants, ΔwbaV mutant showed the highest rate of adhesion (~2.5-fold) and invasion (~4.5-fold) when compared with the wild-type (Fig. 3a, b) and the complemented strain showed adhesion and invasion similar to wild type. However, variations in the efficiency of adhesion and invasion among mutants were observed. The ΔwbaV mutant showed significantly increased adhesion and invasion when compared with other two OAg- negative mutants (P < 0.05–0.001).
Attachment and invasion of S. Enteritidis wild-type and its isogenic mutants to HCT116 cell line. a HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Infection was performed on ice followed by incubation at 4 °C for 30 min. Non adherent bacteria were removed by washing and number of adhering bacteria was enumerated by plating serial dilution. b For invasion, HCT116 cells were seeded at the density of 2 × 105 cells per well and bacterial strains were infected at the MOI of 10. Number of invading bacteria was determined through standard gentamicin protection assay. SPI1 negative strain ΔinvC served as an experimental control. Experiments were performed in triplicates. Percentage of adhesion, invasion and uptake of mutant was compared to wild-type (normalized to 100). Error bar indicate the standard deviation of three independent experiments. Statistical analysis was performed using t test. The percentage of adhesion and invasion replication of Δprt and Δtyv was compared with that of ΔwbaV and level of significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
S. Enteritidis ΔwbaV mutant showed increased systemic load as compared to Δprt mutant in C57BL/6 mice
To understand the effect of deletion of above genes on virulence of ΔwbaV mutant in vivo, C57BL/6 mice were infected with wild-type, ΔwbaV and Δprt mutants. In a 3 days p. i. experiment, the cecal colonization of ΔwbaV mutant was comparable to wild-type, whereas the Δprt mutant colonized less efficiently than ΔwbaV mutant and the wild-type (Fig. 4a), however, the differences were not statistically significant. On the contrary, the pathogen load in the mesenteric lymph node (mLN) varied between both the mutants. The CFU of ΔwbaV mutant recovered from mLN was significantly (P = 0.0085) higher than Δprt mutant (Fig. 4b). Similarly, the bacterial load in spleen and liver was also found to be significantly higher in case of ΔwbaV mutant as compared to Δprt mutant (P < 0.05) (Fig. 4c, d). Notably, the ΔwbaV mutant showed reduced colonization in mLN, spleen and liver as compared to the wild-type. Further, histopathological evaluation was carried out to compare the cecal inflammation between the mutants and wild-type. The ΔwbaV mutant was found to induce moderate cecal inflammation whereas Δprt mutant was unable to induce inflammation in the C57BL/6 mice (Fig. 5a). The cecal pathology was assessed by a semi-quantitative scoring method as described in materials and methods. The day 3 p. i. experiment showed a significant difference in the cecal pathology of the mice infected with Δprt mutant (pathoscore: 8) and ΔwbaV mutant (pathoscore: 2) as compared to the wild-type (P < 0.01) (Fig. 5b). The phenotype of complemented ΔwbaV strain was similar to wild type.
Colonization of cecum, mLN, spleen and liver of C57BL/6 mouse by S. Enteritidis and OAg-negative mutants: Streptomycin pretreated mice were infected with respective bacterial strains through oral route of administration. Mice were sacrificed on 3 days p. i. and cecum (a), mLN (b), spleen (c) and liver (d) were collected and CFU in each organ were determined. Statistical significance is indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), ns not significant
Histopathological evaluation of cecum from mouse infected with S. Enteritidis and OAg-negative mutants. HE stained sections of cryoembeded cecal tissue showing cecal inflammation (a) and respective pathoscore (b) was determined by semi quantitative method as described in "Methods". S submucosal edema, LP lamina propria, L lumen. Bars 200 μm
LPS forms the major component of the outer membrane of Gram-negative bacteria, though ubiquitously present, its structure and composition varies among species. The most variable part of LPS is the OAg chain, consisting of repeating unit of four to six sugars which forms the basis of serological typing. The serovars of Salmonella mainly differ in the presence of dideoxy sugars in OAg repeating unit. In the present study, we have investigated the effect of deletion of three OAg biosynthetic genes on virulence of S. Enteritidis.
Deletion of each gene targeted in this study resulted in the production of OAg-negative LPS. Deletion of prt gene blocks the synthesis of CDP-paratose which cannot be further epimerized to tyvelose by tyv gene product; as a result Δprt mutant cannot synthesize OAg. However, in Δtyv mutant, epimerization of CDP-paratose into tyvelose was hampered due to deletion of tyv gene but the synthesis of CDP-paratose remains unaffected. It has been reported earlier, that tyvelosyl transferase of S. durban has an affinity for CDP-paratose and can transfer it to OAg [10]. Similarly, the group A, B and D glycosyl transferases are able to transfer paratose, abequose and tyvelose to OA [19, 20]. However, Osborn and Weiner, 1968 reported that glycosyl transferase of B1 has more specificity for its natural substrate [21]. In this study, the ability to synthesize complete OAg was restored after complementation indicating that rough phenotype of Δtyv mutant was not due to polar effect. Therefore, the inability of Δtyv mutant to synthesize complete OAg suggests that the tyvelosyl transferase (wbaV) of S. Enteritidis is unable to transfer paratose to OAg repeating unit. Further, deletion of wbaV gene also resulted in the synthesis of OAg-negative LPS, suggesting that, the addition of dideoxy sugar to the OAg sugar backbone is crucial for the complete biosynthesis of OAg. Similar finding was reported by Hong et al. [22].
It is already established in previous studies, that OAg-negative mutants are more susceptible to killing by serum [23, 24], and cationic AMPs such as polymyxin B [23–25]. We also reported the similar findings, and showed that all the mutants were more susceptible to killing by serum and AMPs as compared to wild-type. Earlier reports also demonstrated that the OAg-negative strains have increased efficiency of adhesion and invasion into non phagocytic cells [25, 26]. Our data correlates well with these findings where all the OAg-negative mutants were adhering and invading more efficiently into HCT-116 cells as compared to the wild-type. It was reported that the outer core sugars of LPS are crucial for the interaction of the bacteria with the epithelial cells, [27, 28] therefore, in the absence of OAg, early interaction of core sugars with epithelial cells is facilitated contributing to increased adhesion. Further, increased invasion of O-Ag negative mutants can be explained on the basis of the model proposed by Holzer et al. [25]. The model states that long and very long OAg interferes with the interaction of type III secretion complex with the host cell membrane and impairs the translocation of effector proteins from bacteria to host cell [25]. Thus, in the absence of OAg, this interference is abolished resulting in the increased efficiency of invasion. In this study, mutants showing increased invasion also showed increased adhesion. Therefore, we propose that increased adhesion efficiency along with the enhanced type III secretion system functionality contributed to the significantly increased invasion efficiency to OAg-negative mutants. Notably, in spite of increased invasiveness, the O-Ag negative mutants did not show increased colonization of cecum suggesting, invasiveness alone does not determine the colonization capacity of Salmonella.
In the present study, the ΔwbaV mutant showed increased resistance to AMPs and serum as compared to other OAg-negative mutants. It also showed increased adhesion and invasion as compared to other OAg-negative mutants. To support the in vitro data, mouse experiments were carried out with wild-type, ΔwbaV and Δprt mutants of S. Enteritidis. In, in vivo experiments, no significant difference was observed in cecal colonization of both the mutants, however, the bacterial count of ΔwbaV mutant in mLN was significantly higher than the Δprt mutant. Similarly, ΔwbaV mutant showed increased colonization in spleen and liver as compared to Δprt mutant. Further, ΔwbaV mutant elicited colitis while Δprt mutant was unable to cause colitis. These findings suggest that ΔwbaV mutant is less attenuated as compared to Δprt mutant, which can be explained on the basis of its increased resistance to AMPs and serum.
All the mutants in the present study showed phenotypes similar to an OAg negative strains which are generally avirulent [26]. However, our in vitro and in vivo data demonstrates that the ΔwbaV mutant, in spite of being OAg negative, showed a virulent phenotype. Similar finding was reported by Ilg et al. [26] where mutant of S. Typhimurium lacking O-Ag side chains elicited acute intestinal inflammation. The ability to cause colitis is determined by several factors including stimulation of host immune response which depends on the interaction of LPS and other pathogenesis related patterns with host cell receptors. Modification of LPS is one of the mechanism through which bacteria attain resistance to antimicrobial peptides. Further, LPS modification also affects the interaction of bacteria with TLRs affecting the signaling pathways which are responsible for production of inflammatory cytokines. Recently, Spahich et al. [29] highlighted an important relation between LPS biosynthesis and outer membrane composition [29]. They found that deletion of specific LPS biosynthetic genes downregulates the transcription of an outer membrane protein, Hap and block its insertion into outer membrane [29]. Further, their study also demonstrated that glycosyltransferase activity but not LPS structure affected the insertion of Hap into outer membrane. Their finding suggests that deletion of LPS biosynthetic genes may affect gene expression through unknown mechanism. According to Tam and Missiaka [30], the intermediate compounds of LPS biosynthesis may exert feedback control on LPS biosynthetic pathway which is regulated by σE response. σE belongs to the family of extracytoplasmic factors and serves as an alternative sigma factor encoded by rpoE gene. Earlier studies have reported that changes in outer membrane protein as well as lipopolysaccharide structure induce σE dependent response [30, 31]. In Escherichia coli, σE factor is also involved in transcription of several genes of LPS biosynthetic pathway (for example, rfaD, lpxD, lpxA and ecfA) [32]. rpoE gene is also known to control the alteration in the structure of both lipid A and core oligosaccharide [33]. LPS remodeling has been depicted as a survival strategy employed by bacteria for adaptation to various environmental changes [34]. In present study, the gene wbaV is a glycosyltransferase and deletion of which is expected to result in the accumulation of CDP-tyvelose (intermediate sugar) in the cytoplasm. Also, the ∆wbaV mutant showed increased resistance to antimicrobial peptides and serum which most commonly is observed in case of LPS modifications particularly in lipidA modification [18, 35–37]. Based on the phenotype observed in ∆wbaV mutant, it can be hypothesized that deletion of wbaV gene not only affects the OAg biosynthesis but also certain other unknown cellular process which contributes to the increased resistance to antimicrobial peptides and decreased attenuation in mice.
To conclude, we found that the deletion of wbaV gene conferred an adaptive resistance to antimicrobial peptides resulting in less attenuation in mice as compared to other OAg-negative mutants. Present study demonstrates that certain OAg negative strains can trigger colitis in mouse. Thus, increased resistance to AMPs might be one of the mechanisms employed by S. Enteritidis to compensate the loss of OAg side chains for retaining its pathogenicity.
Bacterial strains and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Wild-type S. Enteritidis P125109 strain and its isogenic mutants were grown in Luria–Bertani (LB) medium (10 g/l Bacto tryptone, 5 g/l Bacto yeast extract, 5 g/l NaCl, HiMedia, India), at 37 °C. For invasion experiments, bacterial strains were grown in SPI1 inducing environment (LB containing 0.3 M NaCl) at 120 rpm. Antibiotics in growth media were used at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; and streptomycin, 50 µg/ml.
Generation of deletion mutant and complementation
Gene deletion was performed using one-step inactivation method as described previously [38]. Briefly, the Tn5 neomycin phosphotransferase gene (aphT) (conferring resistance to kanamycin) in template plasmid pKD4 was PCR amplified using primers listed in Table 2. The primers were designed such that the overlapping coding sequences of adjacent genes do not get deleted and thus the expression of adjacent genes was not affected. The deleted regions have been shown in Fig. 1c. S. Enteritidis wild-type strain harboring plasmid pKD46 was transformed with PCR amplified product and transformants were screened on LB agar plates supplemented with kanamycin. Gene knock-out was confirmed by PCR using kanamycin internal primer and gene specific primers listed in Table 2. For complementation, the respective genes along with 600-bp upstream sequence were cloned between NcoI and XbaI site of plasmid pCH112 and transformed into the corresponding mutant strain [39]. Primers used for complementation have been listed in Table 2.
Table 2 Primers used in this study
LPS analysis
LPS was prepared as described previously [40]. Briefly, bacterial cultures collected from LB agar plate were suspended in phosphate-buffered saline (PBS) with the help of cotton swab and optical density at 600 nm was adjusted to 2.0. From this 1.5 ml suspension was centrifuged and the pellet was resuspended in lysis buffer and boiled at 100 °C for 10 min followed by treatment with proteinase K enzyme and extraction in hot phenol. Final extraction of the aqueous phase was carried out with Tris-saturated ether. LPS preparations were resolved on 14 % acrylamide gels using Tricine-SDS buffer system and visualized by silver staining method as described earlier [40].
Motility assay
Motility of wild-type S. Enteritidis and its isogenic mutants was analyzed by soft agar assay. Briefly, 1 µl of overnight grown culture of S. Enteritidis wild-type and its isogenic mutants was placed at the centre of the soft agar (0.3 % agar) plates and incubated at 37 °C. The diameter of the halo zone was measured after 4 h and 8 h. Plates were photographed with Nikon camera COOLPIXL810. Experiments were performed in triplicates and repeated thrice.
Analysis of resistance to antimicrobial peptides
Overnight grown culture of wild-type and mutant strains was subcultured for 4 h. Cells were washed with PBS and diluted to approximately 108 CFU/ml. A 50 µl aliquot of bacterial suspension was incubated at 37 °C for 1 h with or without AMPs, polymyxin B; 1 µg/ml (Sigma), protamine; 5 µg/ml (Himedia, India), LL37; 10 µg/ml (Sigma), cecropin; 2 µg/ml (Sigma) in PBS. Subsequently, samples were serially diluted and plated on LB agar plates to obtain the number of surviving bacteria. The survival efficiency was calculated as percent ratio of the CFU obtained from AMP treated culture versus untreated culture. Experiment was repeated thrice in triplicates.
Analysis of serum resistance
Human serum samples, devoid of antibody against Salmonella, were obtained from healthy volunteers and pooled, divided into small aliquots and stored at −80 °C. Bacterial cultures grown overnight in LB medium were subcultured for 4 h. Cells were washed with PBS and diluted to approximately 108 CFU/ml. 50 µl of diluted cell suspension was incubated with 50 µl of serum at 37 °C for 1 h. To determine the survival percentage, equal bacterial densities were incubated without serum under similar conditions. Number of surviving bacteria was evaluated by plating serial dilutions on LB agar plate. Survival percentage was calculated by dividing the number of bacteria survived with serum by the number of bacteria obtained from untreated culture. Experiment was performed thrice in triplicates.
Attachment and invasion assay
For attachment and invasion assay, human colonic epithelial cell line (HCT-116) was cultured in Roswell Park Memorial Institute Medium (RPMI 1640) (HiMedia, India) supplemented with 10 % fetal bovine serum (FBS) (HiMedia, India). Cells were seeded on 24 well plates at the density of 2 × 105 cells 16 h prior to infection. For infection, bacterial cultures were grown overnight under SPI1 inducing environment at 120 rpm. Overnight grown culture was diluted to 1:20 in fresh LB and subcultured without antibiotics for 4 h in SPI1 inducing condition. Bacterial cells were washed with PBS and inoculum was prepared in RPMI without antibiotics such that each well received 1 × 106 bacterial cells to achieve a multiplication of infection (MOI) equal to 10. For the attachment assay, both inoculum and cells were prechilled on ice for 15 min to prevent the invasion. Infection was carried out on ice and further incubated for 30 min at 4 °C. Invasion assay was performed as described earlier [41]. Briefly, after 50 min of infection at room temperature, media was replaced with fresh infection media containing 100 µg/ml gentamicin to kill extracellular bacteria and incubated at 37 °C for 2 h in CO2 incubator (5 %). For both the assays, cells were lysed with 0.1 % sodium deoxycholate and appropriate dilutions were plated on LB agar plates supplemented with appropriate antibiotic/s to obtain the adherent and invasive bacteria. The percent of adherent/invasive bacteria was calculated by dividing the number of adherent/invasive bacteria by the number of infecting bacteria and multiplying by 100. Both, adhesion and invasion assays were performed thrice in triplicates.
Mice infection experiments
All animals (C57BL/6 mice) used for the experiments were specific pathogen free (SPF). This animal model is used because of its susceptibility to many broad and narrow host range Salmonella species [42]. Mice were purchased and maintained at the animal house facility of KIIT School of Biotechnology, Odisha, India. 6–8 weeks old specific pathogen free mice were used. All experiments were performed in according to the guidelines of the Institutional Animal Ethics Committee (IAEC), KIIT University, under approval number: KSBT/IAEC/2013/MEET1/A6. During the experiments, all mice were maintained in individually ventilated cages. All the animal experiments were performed in strict accordance with guidelines laid by the Institutional Animal Ethics Committee (IAEC). The infection experiments were carried out in streptomycin pre-treated mice models as established previously [43]. Briefly, a group of five mice were infected (by gavage) with approximately 107 CFU of wild-type, ΔwbaV and Δprt mutants of S. Enteritidis. The bacterial loads in the cecum content, mesenteric lymph node (mLN), spleen and liver were determined at 3 days p. i. by plating suitable dilutions of tissue homogenates on MacConkey agar supplemented with the appropriate antibiotics. For statistical analysis, samples without bacterial counts were adjusted to the minimal detection level (20 CFU/organ in the spleen and 60 CFU/organ in liver). Cecal inflammation of the infected mice was scored to analyze the degree of inflammation.
Histopathological evaluation
Segments of the cecum, colon and ileum were embedded in O.C.T. (Sakura Finetek Inc., USA), snap-frozen in liquid nitrogen, and stored at −80 °C. The 5 μm thick tissue sections were obtained on glass slides and stained with hematoxylin and eosin (H&E) after drying for at least 2 h at room temperature. The stained cryosections were evaluated on the basis of a previously described scoring system for the quantitative analysis of cecal inflammation [43, 44]. The stained sections were scored on the basis of the pathological changes that include sub-mucosal edema, polymorphonuclear leukocyte (PMN) infiltration, loss of goblet cells and epithelial ulceration. The collective pathological score ranges from 0 to 13 with arbitrary units covering the inflammation levels that included intact intestine without any sign of inflammation (pathoscore 0); least sign of inflammation (pathoscore 1–2), slight inflammation as a minimal sign of tissue pathology (pathoscore 3–4); moderate inflammation (pathoscore 5–8); and significant inflammation (pathoscore 9–13).
All the in vitro data represent mean ± standard deviation of three independent experiments. Two-way analysis of variance (ANOVA) was employed to determine significant differences in case of sensitivity to different antimicrobial peptides whereas in remaining experiments t test was used. All the statistical calculations were performed with the help of GraphPad Prism software version 5.
S. Enteritidis:
Salmonella enterica serovar Enteritidis
OAg:
O-antigen
mLN:
mesenteric lymph node
AMP:
antimicrobial peptide
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MS and SJ designed the experiment. SJ, MD, CP, PKS, SR, AA and NKM, performed experiment. NBP performed in vivo experiment. SJ and MS wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by the grant from Department of Biotechnology, Govt. of India (Project No. BT/PR7515/MED/29/673/2012). The research fellowship awarded by "Council of Scientific and Industrial Research (CSIR), New Delhi, India" to Ms. Sangeeta Jaiswal is greatly acknowledged.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
KIIT School of Biotechnology, KIIT University, Bhubaneswar, Odisha, 751024, India
Sangeeta Jaiswal, Niladri Bhusan Pati, Manupriyam Dubey, Chandrashekhar Padhi, Prakash Kumar Sahoo, Shilpa Ray, Aryashree Arunima & Mrutyunjay Suar
Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar, Odisha, 751024, India
Nirmal Kumar Mohakud
Sangeeta Jaiswal
Niladri Bhusan Pati
Manupriyam Dubey
Chandrashekhar Padhi
Prakash Kumar Sahoo
Shilpa Ray
Aryashree Arunima
Mrutyunjay Suar
Correspondence to Mrutyunjay Suar.
Jaiswal, S., Pati, N.B., Dubey, M. et al. The O-antigen negative ∆wbaV mutant of Salmonella enterica serovar Enteritidis shows adaptive resistance to antimicrobial peptides and elicits colitis in streptomycin pretreated mouse model. Gut Pathog 7, 24 (2015). https://doi.org/10.1186/s13099-015-0070-4
S. Enteritidis
OAg-negative
wbaV gene
Antimicrobial peptides
OAg biosynthesis
Submission enquiries: Ayesha.Siddiqka@springernature.com


2019 Annual Meeting - Registration Open!
62nd Annual Scientific Meeting Registration is OPEN!
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SESPRS Family Package includes member, spouse, and all children under 21. Family package includes Welcome Reception, Theme Dinner, and Breakfast. Black Tie is included for member and spouse ONLY, children 16-20 additional black tie fee applies.


depending on the files on src/ for dnd.
dnd files, which you can guess it's wrong.
now nautilus being a library make distcheck stop working.
in the private library to need to access to all dnd functions.
split, which provides more problems than solutions.
it provides, and also, this not so good logical split.
Thanks a lot to Rafael Fonseca for helping in get this done.


Below we have listed a number of activities that are simple, fun and kids just love them! Sharron wants to provide you with resources you can use everyday. Check back for new activities!
This game is a good icebreaker or get-acquainted activity.
Involve movement and counting in this active game.
Ice Sculptures are a really "cool" way to explore water in all its physical states.
Beat the heat with this cool game.


text:
Wind power in Lithuania is a form of renewable energy in Lithuania. At the end of 2011, wind power capacity in Lithuania was 179 MW and the wind energy share of total electricity consumption was 3,8%. At the end of 2019, wind power capacity in Lithuania was 546 MW and the wind energy share of total electricity consumption was 13%.

Statistics
Installed wind power capacity in Lithuania and generation in recent years is shown in the table below:

See also
 Energy in Lithuania
 Renewable energy in Lithuania
 Solar power in Lithuania
 Renewable energy by country

References

External links

Lithuania

redpajama_set_name: RedPajamaWikipedia


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One of the world's truly untamed frontiers, Africa is a rugged land teeming with natural history, phenomenal wildlife and esoteric tribal culture. A world apart from the beaten tourist trails of the western world, Africa stands alone in its capacity to deliver the kind of thrills that inspire sheer adulation towards the natural world.
A continent that's never afraid to speak its mind, North America is big, bold and full of surprises. As LA's epic urban sprawl and New York's monstrous metropolis give way to blockbuster national parks and star-spangled southern towns, this is a destination stuffed full of essential experiences and mind bending must-sees.
Wild and windswept yet far from being a wasteland, Antarctica's sheets of white are an alluring blend of everything and nothingness. With few human inhabitants and wildlife unperturbed by human contact, visitors to Antarctica often feel like ghosts drifting across the Southern Ocean to a land that has no need for the vestiges of human civilisation.
The Australia/Pacific region balances timeless landscapes and ancient cultures with its own keen sense of modernity. With places like the warm, turquoise waters and pristine beaches of the Pacific; dramatic snow-capped peaks and secluded coves of New Zealand; endless red deserts and coastlines of Australia; and the verdant rainforests and charming people of Papua New Guinea.
Colourful, candid and everything in between, South America beats to the rhythm of an infectious, eternal drum. Captured in the chaos of Rio's Carnival, echoing on Columbia's Caribbean beaches and booming along the peaks of the Andes, an effervescent spirit bursts from every facet of life on this remarkable continent.
If you've got no real idea of which style of trip will suit you best, take a look at Intrepid Travel's different trip themes below, as there's something to suit everyone. Whether you're chasing a global gastronomic dream or wanting to ride your bike to Basecamp, Intrepid Travel has a themed adventure that's perfect for you.
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1. What's the probable relationship between the two speakers?
A. Teacher and student.	B. Mother and son.	C. Boss and employee.
2. How often does Bus No.3 run?
A. Every 10 minutes. B. Every 15 minutes.	C. Every 20 minutes.
A. Time management. B. The friendship.	C. TV programs.
4. What does the woman want to do?
A. Go to the downtown market.
B. Go to the department store.
C. Pay a visit to the tower a block away.
5. Where did the man lose his money?
A. In a bank.	B. In a shop.	C. In a hospital.
6. What does the man think is urgent? ? A. Buying some furniture. ?	B. Reducing the budget.????C. Moving into their new home.
7. What does the woman suggest buying?
A. A dining table. B. A bed.	C. A desk.
8. What does the man want to do?
? A. To work on night shift.
? B. To send Steven to school.
? C. To look after Steven.
9. Who should plan Steven's future in the woman's opinion?
A. The father.	B. The mother.	C. Steven himself.
10. What is the woman going to do?
A. Go on a trip.
C. Take part in an interview.
11. What is the woman's major?
A. History.	B. English.	C. Geography.
12. What does the man think of Grace's skirt?
A. It matches her skin well.
B. It's inappropriate for her today.
C. It's sure to attract others' attention.
13. How often does the woman plan to go to the gym?
A. Once a week.	B. Twice a week.	C. Three times a week.
14. What does the woman work out for?
15. What does the man usually do now?
A. He usually plays baseball.
B. He usually watches games on TV.
C. He usually plays tennis with friends.
16. What activity are the speakers both engaged in?
A. Swimming.	B. Playing basketball.	C. Walking.
17. How did a North American Indian boy prove himself an adult?
A. By drinking enough alcohol.
B. By wearing traditional clothes.
C. By staying in the woods alone safely for some time.
18. At what age does an American child legally become an adult?
A. 16.	B. 18.	C. 21.
19. What should the students do after this class?
A. Go online and find more examples of turning into an adult.
B. Summarize the main idea of the class.
C. Prepare their own speech and present it in the next class.
B. Legal activities for adults.
C. Adult ages in different countries.
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The best buys in Rio!
330 rooms in total, a restaurant with regional dishes, parking (additional charge applies), free Wi-Fi, strictly non-smoking, pet allowed.
It's a holiday home allowing bringing your pets in! The bargain!
Free Wi-Fi, a TV, a patio (露台), a self-cooking kitchen with an oven, cash only, 8 persons at most.
21. The four places all provide the service or facilities of ___________.
22. What do we know about Ibis RJ Santos Dumont?
A. It is the only place allowing taking pets in.
B. It is the only place providing the service of Brazilian dishes.
C. It is the nearest to the Gale?o International Airport.
D. It is the closest to the beach.
23. If a rich family wants to enjoy French dishes and sunbathing on the beach, they may ___________.
24. Which might be your best choice if you and your seven poor roommates are interested in sports?
Yesterday mother and I were watching for a cab in the rainy street without an umbrella when an old gentleman sheltering under an umbrella approached us to ask a favor.
Mother began to melt. The idea of getting an umbrella to shelter must have tempted her a good deal. But she didn't want to take advantage of a tired old man in this way. So mother simply gave him the fare but he insisted we take the umbrella. Then the transaction was made and everyone was happy.
As mother was proudly explaining the importance of correctly judging people, I noticed that the old man quickly crossed the street and was hurrying away, not looking tired!
Mother was unhappy. So we followed him as he rushed until we eventually found him at a pub where he ordered a glass of whisky with the pound and swallowed in one go! Then he collected his coat and hat. Just before he left, he smoothly took a wet umbrella from the coat rack (衣帽架). Then he headed back to the main street and sold the umbrella to another unsuspecting person.
25. Why did mother ask the man so many questions before giving him the money?
A. Because mother was just joking with him.
B. Because mother was not a kind-hearted person.
C. Because mother liked him and would like to talk more with him.
D. Because mother wanted to satisfy herself that he was telling the truth.
26. What did the man really do with the money at last?
A. He spent it on alcohol.	B. He kept it to himself.
C. He took a taxi with it.	D. He bought another umbrella with it.
27. Which word may best describe the man in the text?
28. What lesson can we learn from the story?
A. A friend in need is a friend in deed.
B. Kindness is the sunshine of social life.
C. Don't judge a book only by its cover.
D. Think twice before you jump.
A joint research project by researchers from several British universities and institutes has attracted people's attention to the toothbrush again.
Researchers have found that your toothbrush is home to more than 100 million bacteria. But there are hundreds of microorganisms in the mouths and your toothbrush probably won't make you sick unless there is an unhealthy balance of bacteria. Our immune system can usually take care of bacterial invaders. However, if others use your toothbrush, or you use someone else's, bacteria can be spread.
They have also found the toothbrush holders are the third-most dirty household items (behind dish sponges and kitchen sinks), thus advocating cleaning the toothbrush holder regularly.