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Background The efficacy of bariatric surgery may be in part attributed to altered metabolism via new gut microbiome. Milkfat may promote the growth of microbes that are beneficial in long-term weight loss. Understanding the specific gut microbiome changes after surgery and their relationship to milkfat consumption may yield important strategies for managing obesity after bariatric procedures. Methods In this pilot study, stool samples were collected from nine patients before and at the time of surgery, and at 1, 3, and 6 months post-surgery. At each time-point, dairy consumption was determined from dietary surveys. 16 s rRNA gene sequencing was performed followed by alpha diversity analysis. Comparisons of relative abundances of microbial taxa and analyses of fatty acids changes were performed. Results Bariatric surgery led to enrichment of (i) Roseburia, associated with weight loss and (ii) Christensenellaceae, inversely related to body mass index. High milk-fat consumption correlated with enrichment of Blautia, inversely associated with visceral fat accumulation. Faecalibacterium, possibly associated with obesity, increased in patients with low milk-fat consumption. Butter was associated with decreased alpha diversity in all subjects (p-value = 0.038) and the frequency of its use was associated with decreased alpha diversity in patients (correlation = − 0.68, p-value = 0.042). Low-milk-fat consumers showed higher concentration of saturated fatty acids. Conclusions Our results suggest that incorporating dairy products in post-bariatric-surgery dietary plans may help cultivate a gut microbiome that is effective in regulating fat storage as well as digesting beneficial metabolites. These observations will be helpful for the management of obesity in general population as well. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11695-021-05805-z.

The rising prevalence of inflammatory bowel disease (IBD) necessitates that patients be given increased access to cost-effective interventions to manage the disease. Exercise is a non-pharmacologic intervention that advantageously affects clinical aspects of IBD, including disease activity, immune competency, inflammation, quality of life, fatigue, and psychological factors. It is well established that exercise performed at low-to-moderate intensity across different modalities manifests many of these diseased-related benefits while also ensuring patient safety. Much less is known about higher-intensity exercise. The aim of this review is to summarize findings on the relationship between strenuous exercise and IBD-related outcomes. In healthy adults, prolonged strenuous exercise may unfavorably alter a variety of gastrointestinal (GI) parameters including permeability, blood flow, motility, and neuro-endocrine changes. These intensity- and gut-specific changes are hypothesized to worsen IBD-related clinical presentations such as diarrhea, GI bleeding, and colonic inflammation. Despite this, there also exists the evidence that higher-intensity exercise may positively influence microbiome as well as alter the inflammatory and immunomodulatory changes seen with IBD. Our findings recognize that safety for IBD patients doing prolonged strenuous exercise is no more compromised than those doing lower-intensity work. Safety with prolonged, strenuous exercise may be achieved with adjustments including adequate hydration, nutrition, drug avoidance, and careful attention to patient history and symptomatology. Future work is needed to better understand this intensity-dependent relationship so that guidelines can be created for IBD patients wishing to participate in high-intensity exercise or sport.

BackgroundHelicobacter pylori, a bacterium that infects approximately half of the world’s population, is associated with various gastrointestinal diseases, including peptic ulcers, non-ulcer dyspepsia, gastric adenocarcinoma, and gastric lymphoma. To combat the increasing antibiotic resistance of H. pylori, the need for new therapeutic strategies has become more pressing. Characterization of the interactions between H. pylori and the fecal microbiome, as well as the mechanisms underlying these interactions, may offer new therapeutic approaches. Exploration of changes in fatty acid metabolism associated with H. pylori-mediated alterations of the fecal microbiome may also reveal strategies to help prevent progression to neoplasia.AimTo characterize the gut microbiome and metabolome in H. pylori patients in a socioeconomically challenged and underprivileged inner-city community.MethodsStool samples from 19 H. pylori patients and 16 control subjects were analyzed. 16S rRNA gene sequencing was performed on normalized pooled amplicons using the Illumina MiSeq System using a MiSeq reagent kit v2. Alpha and beta diversity analyses were performed in QIIME 2. Non-targeted fatty acid analysis of the samples was carried out using gas chromatography-mass spectrometry (GC-MS), which measures the total content of 30 fatty acids in stool after conversion into their corresponding fatty acid methyl esters. Multi-dimensional scaling (MDS) was performed on Bray-Curtis distance matrices created from both the metabolomics and microbiome datasets and a Procrustes test was performed on the metabolomics and microbiome MDS coordinates.ResultsFecal microbiome analysis showed that alpha diversity was lowest in H. pylori patients over 40 years of age compared to control subjects of similar age group. Beta diversity analysis of the samples revealed significant differences in microbial community structure between H. pylori patients and control subjects. Thirty-eight and six taxa had lower and higher relative abundance in H. pylori patients, respectively. Taxa that were enriched in H. pylori patients included Atopobium, Gemellaceae, Micrococcaceae, Gemellales and Rothia (R. mucilaginosa). Notably, relative abundance of the phylum Verrucomicrobia was decreased in H. pylori patients compared to control subjects, suggesting disruption of the gut mucosal environment by H. pylori. Procrustes analysis showed a significant relationship between the microbiome and metabolome datasets. Stool samples from H. pylori patients showed increases in several fatty acids including the polyunsaturated fatty acids (PUFAs) 22:4n6, 22:5n3, 20:3n6 and 22:2n6, while decreases were noted in other fatty acids including the PUFA 18:3n6. The pattern of changes in fatty acid concentration correlated to the Bacteroidetes:Firmicutes ratio determined by 16S rRNA gene analysis.ConclusionAn individualized understanding of gut microbiome features among H. pylori patients will pave the way for improved community impact, reduced healthcare burdens of repeated treatment, and decreased mounting resistance.

Several communities have started using probiotic-rich fermented foods as therapeutic options with presumed medicinal powers. We now know the importance of microbiome balance and how probiotics can restore imbalances in the microbiome. Probiotics have been tested for a number of clinical uses such as the prevention of antibiotic-associated diarrhea (AAD), the treatment of various diseases such as H. pylori infection, irritable bowel disease, vaginitis, the prevention of allergies, and necrotizing enterocolitis in newborns. AAD has been the most indicated therapeutic use for probiotics. AAD is a common side effect of antibiotic usage, which affects up to 30% of patients. The hypothesis behind using probiotics for AAD is that they help normalize an unbalanced flora. There are many potential mechanisms by which probiotics support intestinal health such as (i) boosting immunity, (ii) increasing gut barrier integrity, (iii) producing antimicrobial substances, (iv) modulating the gut microbiome, (v) increasing water absorption, and (vi) decreasing opportunistic pathogens. Many randomized-controlled trials including the strain-specific trials that use Lactobacillus and Saccharomyces and meta-analyses have shown the benefits of probiotics in addressing AAD. Although adverse events have been reported for probiotics, these are broadly considered to be a safe and inexpensive preventative treatment option for AAD and other gastrointestinal disorders.

Reports of adverse effects associated with proton pump inhibitors (PPIs) are concerning because of high usage and over-the-counter availability. We sought to determine the awareness of PPI adverse effects among our patient population, which is medically underserved, low-income, and racially diverse. A 21-item survey was administered to gastroenterology-clinic outpatients. It collected information about age, gender, education, race, specialty of the prescriber, specific PPI, indication, knowledge of dose, adherence, duration of use and awareness of any risks. Medical records were reviewed to verify survey responses pertaining to indication, dosing, and adherence. A vast majority (96%) of 101 participants were not aware of PPI adverse effects. In total, 63% of the patients completed a high school education or less, which was associated with a higher risk of long-term PPI use than completion of at least an undergraduate degree (p = 0.05). In contrast to other studies, the shockingly low patient awareness about PPI adverse effects in our patient population is particularly concerning, especially as it is tied to their demographic attributes. This may lead to long-term and high-dose PPI use. Our study highlights the need for effective provider-driven education regarding medication risks, especially in the communities with significant health disparities.

Background Regional variation in Helicobacter pylori resistance patterns is a significant contributing factor for the ineffectiveness of traditional treatments. To improve treatment outcomes, we sought to create an individualized, susceptibility-driven therapeutic approach among our patient population, which is one of the poorest in the nation. It is medically underserved, minority-predominant and has high incidence of H pylori infection. Methods We compiled various factors involved in the antibiotic resistance of H pylori from literature. We then created a predictive model to customize therapies based on analyzed data from 2,014 H pylori patients with respect to several of these factors. The predictions of the model were further tested with analysis of patient stool samples. Results A clear pattern of H pylori prevalence and antibiotic resistance was observed in our patients. We observed that majority of H pylori patients were women (62%) and over the age of 40 years (80%). 30% and 36% of the H pylori patients were African American and Hispanic, respectively. A median household income of less than $54,000, past H pylori infection, previous use of certain antibiotics for any infection decreased the chance of eradication. Results of the stool testing were consistent with model predictions (90% accuracy). Conclusion This model demonstrates the predictive accuracy of H pylori infection and antibiotic resistance based on patient attributes and previous treatment history. It will be useful to formulate customized treatments with predicted outcomes to minimize failures. Our community attributes may contribute toward broad applicability of model for other similar communities.

BACKGROUND: Integrating basic science into clinical teaching has been a struggle for medical schools. However, early exposure to clinical experience has been associated with an increased understanding of the importance of basic science, positive attitudes, and developing clinical skills faster. Furthermore, early clinical exposure can help students reconnect with what drove them into medicine in the first place, especially when they are starting to feel burned out by the volume of lecture material. As a result, increasing patient experience during the first year has become a goal of many medical schools. METHODS: At Rutgers Robert Wood Johnson Medical School, interprofessional case discussions (ICDs) begin with a lecture that explicitly integrates basic science with a disease, followed by a discussion with a patient, their family, the healthcare team, and first-year students. Our objective is to explore whether ICDs enhanced the learning experience of basic science. CONTEXT: ICD satisfaction was assessed using evaluations from two different courses (2013–2016). Responses were analyzed quantitatively using descriptive statistics and qualitatively using a grounded-theory-content analysis. Study 2: A follow-up measure with current third- and fourth-year students on long-term retention of basic science was analyzed using a Wilcoxon signed rank test. Relative rankings of three different case-based teaching modalities were assessed using chi-square. RESULTS: Students reported significantly higher satisfaction with ICDs (93%) for reinforcing concepts and integrating materials compared to Flipped Classrooms (66%) and Jigsaws (65%), x(2) = 120.9, p

Learner-centered pedagogical methods that are based on clinical application of basic science concepts through active learning and problem solving are shown to be effective for improving knowledge retention. As the clinical relevance of biochemistry is not always apparent to health-profession students, effective teaching of medical biochemistry should highlight the implications of biochemical concepts in pathology, minimize memorization, and make the concepts memorable for long-term retention. Here, we report the creation and successful implementation of a flipped jigsaw activity that was developed to stimulate interest in learning biochemistry among medical students. The activity combined the elements of a flipped classroom for learning concepts followed by a jigsaw activity to retrieve these concepts by solving clinical cases, answering case-based questions, and creating concept maps. The students' reception of the activity was very positive. They commented that the activity provided them an opportunity to review and synthesize information, helped to gage their learning by applying this information and work with peers. Students' improved performance especially for answering the comprehension-based questions correctly in the postquiz as well as the depth of information included in the postquiz concept maps suggested that the activity helped them to understand how different clinical scenarios develop owing to deviations in basic biochemical pathways. Although this activity was created for medical students, the format of this activity can also be useful for other health-professional students as well as undergraduate and graduate students. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:343-353, 2018.

A number of active learning approaches are being used in medical schools and are shown to be effective for improving knowledge comprehension and retention. Here, we report the creation and successful implementation of an interactive activity that was created to help students to understand the concepts of anatomy. Our goal was to encourage them to think about human structure and function by demonstrating how anatomy is changed due to disease or trauma. This session consolidated clinically relevant concepts from different anatomy didactic sessions into one exercise. It also allowed application of basic anatomy principles to clinical scenarios by comparing normal thoracic anatomy with the trauma specimens such as those from chylothorax, traumatic aortic injury, superior vena cava (SVC) syndrome, and tracheobronchial injury with pneumomedistinum. The session was received very well by the students and resulted in their improved understanding and application of various principles of anatomy.

The heat shock proteins (Hsp), the family of molecular chaperons, are key proteins in protein folding and maturation. The client proteins of Hsp are critical in number of biological processes including cellular proliferation, differentiation, survival, metastasis, invasion, and angiogenesis. Thus, Hsp family becomes one of the desirable targets for cancer treatment. It has been demonstrated that Hsp overexpress in multiple myeloma and linked in poor prognosis and relapse. This chapter describes about the Hsp and their possible link with the pathogenesis of multiple myeloma. It addresses the advancement and challenges in the development of Hsp inhibitors.

Various studies have emphasized the need to improve the nutrition training of health professionals, which will help them to provide optimal patient care. Nutrition-based interactive sessions may serve as an efficient approach to instigate an interest in nutrition among the students. Here we report the reception and effectiveness of a nutrition-pathology based interactive activity that we designed and implemented in the gastroenterology course given to the second year students at our medical school. The activity involved team work, individual accountability and peer-teaching. Nutrition pathology case stems (Kwashiorkor, vitamin B-12 deficiency, zinc deficiency and zinc-induced copper deficiency) were posted on the course website for the students to read before the session. At the start of the session, all the groups (each made up of four members) took a pre-quiz. Each student was then given an information sheet describing one case. Each group discussed the four cases with students acting as the “teacher” for the case assigned to them. A post-quiz was administered to the groups to assess acquisition of knowledge as well as in-depth thinking about the nutrition aspects discussed. The efficacy of the session measured by pre (39% questions correctly answered in total) and post-quizzes (96% questions correctly answered in total) and the overwhelmingly positive student feedback indicated that the session was highly effective. Ninety-five percent of students thought that the session demonstrated the clinical relevance of nutrition, while 98% students found the peer teaching to be engaging.

Mother Nature has blessed us with a variety of natural compounds rich in anti-inflammatory and anti-tumor properties. In the past three decades, plant-derived natural compounds, popularly referred to as nutraceuticals, have received a lot of attention because they are economic, bioavailable, safe, and lethal to cancers. One of noteworthy nutraceuticals is gambogic acid (GA). GA that is derived from gamboge resins secreted by a plant known as Garcinia hanburyi. Extensive preclinical studies have strongly suggested that GA possesses anti-cancer properties against a variety of malignancies including solid and hematologic cancers. GA exerts its effects on cancer cells through several different mechanisms. Interestingly, normal human cells were found to be resistant to GA, suggesting that this nutraceutical has tremendous therapeutic values. Chemoresistance has been recognized as one of the largest hurdles facing anti-cancer regimens. Therefore, researchers have continued to search for potent chemosensitizers. This chapter focuses on the chemosensitizing properties of GA, mechanisms through which GA exerts anti-proliferative and apoptotic effects in cancer. GA sensitizes acute myeloid leukemia (AML), gastric cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal carcinoma, and multiple myeloma (MM). GA acts synergistically with many chemotherapeutic agents including adriamycin, cisplatin, doxorubicin, 5-fluorouracil (5-FU), gefitinib, and proteasome inhibitors. Overall, in vitro and studies using animal models demonstrate that GA can be used to sensitize tumors to chemotherapy. Nonetheless, a clinical trial is needed in order to fully delineate the utility of this nutraceutical in human cancers.

Learner-centered pedagogical methods that are based on clinical application of basic science concepts through active learning and problem solving are shown to be effective for improving knowledge retention. As the clinical relevance of biochemistry is not always apparent to health-profession students, effective teaching of medical biochemistry should highlight the implications of biochemical concepts in pathology, minimize memorization, and make the concepts memorable for long-term retention. Here, we report the creation and successful implementation of a flipped jigsaw activity that was developed to stimulate interest in learning biochemistry among medical students. The activity combined the elements of a flipped classroom for learning concepts followed by a jigsaw activity to retrieve these concepts by solving clinical cases, answering case-based questions, and creating concept maps. The students' reception of the activity was very positive. They commented that the activity provided them an opportunity to review and synthesize information, helped to gage their learning by applying this information and work with peers. Students' improved performance especially for answering the comprehension-based questions correctly in the postquiz as well as the depth of information included in the postquiz concept maps suggested that the activity helped them to understand how different clinical scenarios develop owing to deviations in basic biochemical pathways. Although this activity was created for medical students, the format of this activity can also be useful for other health-professional students as well as undergraduate and graduate students. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:343-353, 2018.

Reminiscent of eukaryotic apoptotic programmed cell death, bacteria also contain a large number of suicide genes, which are in general co-expressed with their cognate antitoxin genes. These systems called the toxin-antitoxin (TA) systems are associated with cellular dormancy, and play major roles in biofilm formation and persistent multidrug resistance of many human pathogens. In recent years, the study on TA system toxins has become a hot topic due to the health implications of these toxins by virtue of their role in bacterial pathogenicity. Here we report functional characterization of a hitherto uncharacterized Escherichia coli TA toxin, YjjJ. YjjJ exhibits several uncommon properties: (i) unlike the genes encoding most type II TA system toxins, the gene encoding YjjJ is present as a single gene and not in an operon, (ii) despite being a homolog of the well-characterized toxin HipA, YjjJ seems to have different cellular target(s), and (iii) HipB, the cognate antitoxin of HipA, also acts as an antitoxin for YjjJ. This forms a basis for an interesting next step in the study of TA systems with respect to cross-regulation between various TA systems and the evolutionary as well as clinical significance of these observations.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder that is characterized by symptoms of chronic abdominal pain and altered bowel habits in the absence of an overtly identifiable cause. It is the most commonly diagnosed functional gastrointestinal disorder, accounting for about one third of gastroenterology visits. It generally presents as a complex of symptoms, including psychological dysfunction. Hypersensitivity to certain foods, especially foods that contain high amounts of fructose, plays a role in the pathophysiology of IBS. Elevated consumption of high-fructose corn syrup (HFCS) has been discussed in this aspect. The treatment options for IBS are challenging and varied. In addition to dietary restrictions for HFCS-induced IBS, such as low-FODMAP (Fermentable Oligosaccharides, Disaccharide, Monosaccharides, and Polyols) diets, existing drug therapies are administered based on the predominant symptoms and IBS-subtype. Patients with IBS are likely to suffer from issues, such as anxiety, depression, and post-traumatic-stress disorder. Biopsychosocial factors particularly socioeconomic status, sex, and race should, thus, be considered for diagnostic evaluation of patients with IBS.

To promote consistent thinking about nutrition, we gave the students thought-provoking, one-a-day nutrition questions throughout a 4-week gastroenterology course. The interactive and detailed feedback for each question also served as an education tool. The questions helped to increase competency in nutrition by enhancing learning and memory retention of concepts.

The rationale for this mandatory, guided online e-journal exercise is to foster the ability of students to independently read medical and scientific literature in a critical manner and to integrate journal reading with their basic science knowledge. After a lecture on oxidative phosphorylation, students were assigned to read an article on brown adipose tissue published in New England Journal of Medicine and were guided to analyze the article by answering online questions. After two iterations, student surveys about the project, its key pedagogical features, and ways to improve it suggest that the students perceived these exercises as active learning, which is clinically relevant and built on their course material. Furthermore, students agreed that the e-journal project was useful for learning how to read an article, for reviewing the material learned in class, and for promoting evidence-based medicine. This online e-journal exercise models some aspects students will experience as future physicians, where it is essential to keep up with literature and extract relevant information on a tight physician's schedule. This study demonstrated the usefulness of guided e-journal exercises as a simple effective active teaching tool for preclinical medical students, which can also be used for prehealth undergraduate programs.

We designed and implemented several active learning approaches at our medical school to stimulate student interest in nutrition and to help them feel more comfortable and competent in talking about nutrition. Positive feedback and overall engagement of students suggested that these approaches stimulated their interest in basic aspects of nutrition.

Reminiscent of apoptotic genes in higher systems, almost all bacteria contain ‘suicide' genes, which encode toxins. Toxins inhibit cell growth and may cause cell death. These are coexpressed with their cognate antitoxins from a toxin-antitoxin (TA) operon in normally growing cells. The cellular targets of toxins are diverse. The study of the TA system is important as in addition to their involvement in the normal bacterial physiology, TA systems may also play a role in bacterial pathogenicity. The long direct repeat (Ldr) family of proteins belongs to one such TA system. Here we report characterization of LdrA, the prototypical protein of the Ldr family, and show that it is highly toxic for cell growth. The data suggests that LdrA may exert toxicity by inhibiting ATP synthesis, possibly due to its localization in the cell membrane. This inhibition of energy production leads to the inhibition of biosynthetic reactions in the cell such as DNA replication, transcription and translation, and eventually cell growth.

For successful delivery of basic science topics for health-professional students, it is critical to reduce apprehension and illustrate relevance to clinical settings and everyday life. At the beginning of the Biochemistry course for Physician Assistants, a team-based assignment was designed to develop an understanding of the mechanism of action, effectiveness, and toxicity of five common over the counter (OTC) drugs and dietary supplements, and place these familiar medicines in a political and historical context. The objectives of this exercise were to stimulate interest in biochemistry; to provide basic information on enzymes and enzyme inhibitors related to these drugs to be expanded upon later in the course; and to encourage active and interactive learning. Teams of five students were formed, and each student was given an information sheet on aspirin, alpha-galactosidase, orlistat, dextromethorphan, or simvastatin, a low dose statin, which was previously available without prescription at pharmacies in the UK. After each member of the team acquired information on one OTC drug/dietary supplement by reading an assigned information sheet, the team was asked to go through a series of questions, and then submit answers to a quiz as a group. A high rate of success on the quiz, an overwhelmingly positive response on formal course evaluations, and enthusiastic exchanges during class suggested this team-based session accomplished its goals.

Cold-shock response is elicited by the transfer of exponentially growing cells from their optimum temperature to a significantly lower growth temperature and is characterized by the induction of several cold-shock proteins. These proteins, which presumably possess a variety of different activities, are critical for survival and continued growth at low temperature. One of the main consequences of cold shock is stabilization of the secondary structures in nucleic acids leading to hindrance of RNA degradation. Cold-shock proteins, such as RNA helicase CsdA, and 3'-5' processing exoribonucleases, such as PNPase and RNase R, are presumably involved in facilitating the RNA metabolism at low temperature. As a step toward elucidating the individual contributions of these proteins to low-temperature RNA metabolism, the global transcript profiles of cells lacking CsdA, RNase R and PNPase proteins as well as cells individually over-expressing these proteins as compared to the wild-type cells were analyzed at 15 °C. The analysis showed distinct sets of genes, which are possible targets of each of these proteins. This analysis will help further our understanding of the low-temperature RNA metabolism.

Temperature downshift from 37 °C to 15 °C results in the exertion of cold shock response in Escherichia coli, which induces cold shock proteins, such as CsdA. Previously, we showed that the helicase activity of CsdA is critical for its function in the cold acclimation of cells and its primary role is mRNA degradation. Only RhlE (helicase), CspA (RNA chaperone) and RNase R (exoribonuclease) were found to complement the cold shock function of CsdA. RNase R has two independent activities, helicase and ribonuclease, only helicase being essential for the functional complementation of CsdA. Here, we discuss the significance of above findings as these emphasize the importance of the unwinding activity of cold-shock-inducible proteins in the RNA metabolism at low temperature, which may be different than that at 37 °C. It requires assistance of proteins to destabilize the secondary structures in mRNAs that are stabilized upon temperature downshift, hindering the activity of ribonucleases.

One of the many important consequences that temperature down-shift has on cells is stabilization of secondary structures of RNAs. This stabilization has wide-spread effects, such as inhibition of expression of several genes due to termination of their transcription and inefficient RNA degradation that adversely affect cell growth at low temperature. Several cold shock proteins are produced to counteract these effects and thus allow cold acclimatization of the cell. The main RNA modulating cold shock proteins of E. coli can be broadly divided into two categories, (i) the CspA family proteins, which mainly affect the transcription and possibly translation at low temperature through their RNA chaperoning function and (ii) RNA helicases and exoribonucleases that stimulate RNA degradation at low temperature through their RNA unwinding activity.

In recent years, there has been an increasing focus on the need to integrate formal knowledge with clinical experience in the pre-clinical years since the initial years of medical education play an important role in shaping the attitudes of medical students towards medicine and support the development of clinical reasoning. In this study, we describe approaches that involve real patients and patient-simulation-based methodologies to teach gastroenterology to second year medical students. Our goals were to (i) demonstrate bio-psychosocial aspects of clinical practice, (ii) demonstrate commonality of gastrointestinal ailments, and (iii) help understand complex gastroenterology concepts. We used two main approaches including brief, pre-prepared questions and answers discussing with the patients in various sessions throughout the course and a two-hour session that included patient participation, patient simulation modalities with high fidelity mannequins, a lightening round of interactive cases, and a Patient Oriented Problem Solving (POPS) session. The approaches improved the effectiveness of the delivery of the content-heavy, fast-paced GI course and provided opportunities for the students to think about gastroenterology from both basic and clinical points of view. The approaches involved peer teaching, which supports knowledge acquisition and comprehension. Very positive feedback and overall engagement of students suggested that these approaches were well-received.

In Escherichia coli, the cold shock response is exerted upon temperature change from 37 to 15°C and is characterized by induction of several cold shock proteins including its major cold shock protein, CspA. E. coli CspA family consists of nine members, CspA to CspI. CspA and some of its homologues play a critical role in cold acclimation of cells as RNA chaperones by destabilizing secondary structures in RNAs. Here, we showed that the nucleic acid melting activity of Csp proteins can be used to facilitate reactions, such as RT-PCR or RNA cleavage reactions by endoribonucleases, which are hindered by presence of secondary structures in the DNA/RNA substrate used. The low substrate specificity of Csps together with their compatibility with various enzymes and their stability and activity over a broad temperature range makes them ideal candidates to be used for a variety of processes.

In Escherichia coli , the cold shock response is exerted upon a temperature change from 37°C to 15°C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli , PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3′-to-5′ exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3′-to-5′ exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3′-to-5′ processing exoribonuclease, RNase R of E. coli , which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis , in the Δ pnp cells has consequences, such as accumulation of ribosomal subunits in the Δ pnp cells, which may play a role in the cold sensitivity of this strain.

mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to –mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UUˇCCU or CUˇCCU and MazF-mt7 at UˇCGCU (ˇindicates the cleavage site). Since pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression though differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.

The Escherichia coli cold shock protein CspA family consists of nine proteins (CspA to CspI), of which two, CspE and CspC, are constitutively produced at 37°C and are involved in regulation of expression of genes encoding stress response proteins but can also perform an essential function during cold acclimation. In this study, we analyzed global transcript profiles of cells lacking cspE and cspC as well as cells individually overexpressing these proteins or a CspE mutant that is unable to melt nucleic acids and is defective in cold acclimation. The analysis reveals sets of genes whose expression (i) is regulated by CspC and CspE at physiological temperature or cold shock conditions and (ii) depends on the nucleic acid melting function of CspE. Bioinformatic analysis of the latter group reveals that many of those genes contain promoter-proximal sequences that can block transcript elongation and may be targeted by the nucleic acid melting function of CspE.

Escherichia coli contains nine members of the CspA family. CspA and some of its homologues play critical role in cold acclimation of cells by acting as RNA chaperones, destabilizing nucleicacid secondary structures. Disruption of nucleic acid melting activity of CspE led to loss of its transcription antitermination activity and consequently its cold acclimation activity. To date, the melting activity of Csp proteins was studied using partially double-stranded model nucleic acids substrates forming stem–loop structures. Here, we studied the mechanism of nucleic acid melting by CspE. We show that CspE melts the stem region in two directions, that CspE-induced melting does not require the continuity of the substrate's loop region, and CspE can efficiently melt model substrates with single-stranded overhangs as short as 4 nt. We further show that preferential binding of CspE at the stem–loop junction site initiates melting; binding of additional CspE molecules that fully cover the single-stranded region of a melting substrate leads to complete melting of the stem.

A DNA microarray-based global transcript profiling of Escherichia coli in response to cold shock showed that in addition to the known cold shock-inducible genes, new genes such as the flagellar operon, those encoding proteins involved in sugar transport and metabolism, and remarkably, genes encoding certain heat shock proteins are induced by cold shock. In the light of strong reduction in metabolic activity of the cell after temperature downshift, the induction of sugar metabolism machinery is unexpected. The deletion of four csp s ( cspA , cspB , cspG , and cspE ) affected cold shock induction of mostly those genes that are transiently induced in the acclimation phase, emphasizing that CspA homologues are essential in the acclimation phase. Relevance of these findings with respect to the known RNA chaperone function of CspA homologues is discussed.

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

Background: Cold-shock proteins (Csps) are important for cellular adaptation to low temperature. Csps help cells adapt to low-temperature growth through their RNA-binding and nucleic acid melting abilities, which lead to anti-termination of transcription. Results: We studied the two most thermostable Csps known to date, TmCspB and TmCspL from Thermotoga maritima, a hyperthermophilic eubacterium for which no cold-shock response has been demonstrated so far. For comparison, we used a well-characterized Escherichia coli CspE protein. TmCspB and TmCspL are able to bind RNA at both low and high temperatures. They are also able to ‘melt’ nucleic acids secondary structures and as a result decrease E. coli RNA polymerase transcription termination in vivo and E. coli and T. maritima RNA polymerases transcription termination in vitro. Over-expression of TmCsps allowed E. coli cold-sensitive mutant cells to acclimate to the low temperatures of 15 °C. Conclusions: TmCspB and TmCspL (i) are able to perform essential functions of E. coli Csps in vitro and in vivo, 50–65 °C below the temperature optimum of T. maritima and (ii) can anti-terminate transcription by T. maritima RNA polymerase at 55 °C, the lower limit of temperature range for growth of T. maritima. We propose that the observed properties of TmCsps are physiologically relevant and that TmCsps are important for adaptation of T. maritima to physiologically low temperatures.

We carried out DNA microarray-based global transcript profiling of Escherichia coli in response to 4,5-dihydroxy-2-cyclopenten-1-one to explore the manifestation of its antibacterial activity. We show that it has widespread effects in E. coli affecting genes encoding proteins involved in cell metabolism and membrane synthesis and functions. Genes belonging to the regulon involved in synthesis of Cys are upregulated. In addition, rpoS and RpoS-regulated genes responding to various stresses and a number of genes responding to oxidative stress are upregulated.

Members of bacterial Csp (cold-shock protein) family promote cellular adaptation to low temperature and participate in many other aspects of gene expression regulation through mechanisms that are not yet fully elucidated. Csp proteins interact with single-stranded nucleic acids and destabilize nucleic acid secondary structures. Some Csp proteins also act as transcription antiterminators in vivo and in vitro. Here, we selected a mutation in the cloned cspE gene that abolished CspE-induced transcription antitermination. In vitro, mutant CspE showed RNA binding activity similar to that of the wild-type CspE but was unable to destabilize nucleic acid secondary structures. Thus, nucleic acid melting ability of CspE and its transcription antitermination activity are correlated. In vivo, mutant cspE was functional with respect to up-regulation of expression of rpoS, but, unlike the wild-type cspE, it did not complement the cold-sensitive phenotype of the quadruple DeltacspADeltacspBDeltacspGDeltacspE deletion strain. Thus, the nucleic acid-melting activity of Csp is critical for its prototypical function of supporting low temperature survival of the cell.

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.