Consequences of medical negligence and litigations on health care providers - A narrative review.

Medical professionals face high stress due to the type of work they do and the prolonged working hours. Frequent burnout results due to the challenging nature of their work. Added to the stress of work, malpractice lawsuits add to their burden. In India, most doctors work in compromised settings with poor infrastructure and manpower but are expected to follow the best practices. In court, they are judged with the Bolam and Bolitho tests being essential considerations. Several tragic incidents have been reported, including depression, anger issues, and even suicide deaths of healthcare professionals (HCPs) after accusations of negligence and subsequent inquiry. Such incidents demonstrate the multitude of challenges an HCP faces in day-to-day practice. It is crucial to find ways to tackle these problems and enhance the capacity of HCP to handle such demanding circumstances. Malpractice litigation can significantly impact the mental health of HCPs. It is common to experience emotional turmoil when faced with a lawsuit. Second victim syndrome (SVS) is a term used to describe a set of symptoms experienced by HCPs who make an error leading to injury to a patient. However, it also happens if he is traumatized by the consequences of violence during healthcare services or a lawsuit or defamation article in newspaper/social media. Following a litigation crisis in their career, many HCPs go through various stages of grief, including shock, denial, anger, bargaining, depression, and acceptance. At times, death by suicide of the HCPs is well known. SVS is known to profoundly affect the personal, family, economic, professional (defensive practice), and social life of HCPs. HCPs should accept the allegations of negligence as an occupational hazard and prepare for the eventual litigation at least once in a lifetime by knowing about the medical laws, HCP's rights, becoming aware of the emotional turmoil of the lawsuit, preparing to cope with the lawsuit, and seeking help from colleagues and indemnity insurance. Frequent training of the HCPs is strongly recommended to know about the changing laws and also to undergo periodic professional competence enhancement to reduce the incidents of errors amounting to medical negligence. Medical and hospital administration should debrief after any incident and conduct internal investigations to identify systemic flaws and prevent future recurrence, resolve issues within their control at their level, and manage media (mainstream and social media) appropriately. If established, a reporting system with online and offline services will ease the internal administrative investigation process and take appropriate, timely actions. During the crisis, HCPs should have adequate and appropriate insurance or indemnity coverage and mental health support systems.

Prospective Phase II Study of Radiotherapy Dose Escalation in Grade 4 Glioma Using 68Ga-Pentixafor PET Scan.

AIMS: Local failure remains the major concern in grade 4 glioma or glioblastoma (GBM). Pilot studies have shown a radiotherapy (RT) dose-response relationship in GBM. Here we present our preliminary data of RT dose escalation using 68Ga-Pentixafor PET scan. High 68Ga-pentixafor uptake in glioma cells helps in sharp demarcation between tumour and normal brain. MATERIALS AND METHODS: This phase II prospective study was conducted from 2018 to 2020. Thirty, biopsy-proven cases of grade 4 glioma were included. All patients underwent post-operative MRI of the brain and 68Ga-Pentixafor PET scan. RT was planned in 2-phases. Phase-1 GTV (GTV1) comprised of T2/flair abnormality, PET-avid disease and post-op cavity. A margin of 2cm was given to GTV-1 to create phase-1 CTV (CTV1), which was further expanded to 0.5cm to generate phase-1 PTV (PTV1). A radiation dose of 46Gy/23fr was prescribed to PTV-1. Phase-2 GTV (GTV2) consisted of CT/MRI contrast-enhancing lesion, PET avid disease and post-op cavity. A margin of 0.5 cm was given to GTV2 to create phase-2 CTV (CTV2) which was expanded to 0.5 cm to create phase-2 PTV (PTV2). RT dose of 14 Gy/7 fr was prescribed to PTV2. PET avid disease was delineated as GTV PET and a margin of 3mm was given to generate PTV-PET which received escalated RT dose of 21 Gy/7fr by simultaneous integrated boost (SIB) in phase 2 (Total dose to PTV PET = 67 Gy/30 fr). All patients received concurrent and adjuvant temozolomide. The data was prospectively maintained in Microsoft Excel sheet. SPSS v 23 was used for statistical analysis. The primary endpoints were estimation of the overall survival (OS) and progression-free survival (PFS), and secondary endpoint was to measure the incidence of radiation necrosis. Categorical variables were reported as frequency and percentage and quantitative variables were reported as median and range. RESULTS: Data from thirty patients were analysed. A median OS of 23 months was observed with estimated 1, 2 and 3 years OS of 90%, 40% and 17.8% respectively. A significant association of OS was seen with the extent of surgery (p = 0.04) and kernofsky performance status (p = 0.007). No patient developed significant radiation necrosis. CONCLUSIONS: The index study did not show any survival benefit from dose escalation RT. However, all of the patients tolerated the treatment well and none of them developed radiation necrosis. Considering the small sample size as a limitation of the index study, the role of 68Ga-pentixafor PET scan for radiation dose escalation should be further explored. CLINICAL TRIAL NUMBER: CTRI/2019/05/019146.

Recent developments in enzymatic and microbial biosynthesis of flavor and fragrance molecules.

Flavors and fragrances are an important class of specialty chemicals for which interest in biomanufacturing has risen during recent years. These naturally occurring compounds are often amenable to biosynthesis using purified enzyme catalysts or metabolically engineered microbial cells in fermentation processes. In this review, we provide a brief overview of the categories of molecules that have received the greatest interest, both academically and industrially, by examining scholarly publications as well as patent literature. Overall, we seek to highlight innovations in the key reaction steps and microbial hosts used in flavor and fragrance manufacturing.

Harnessing biocatalysis to achieve selective functional group interconversion of monomers.

Polymeric materials are ubiquitous to modern life. However, reliance of petroleum for polymeric building blocks is not sustainable. The synthesis of macromolecules from recalcitrant polymer waste feedstocks, such as plastic waste and lignocellulosic biomass, presents an opportunity to bypass the use of petroleum-based feedstocks. However, the deconstruction and transformation of these alternative feedstocks remained limited until recently. Herein, we highlight examples of monomers liberated from the deconstruction of recalcitrant polymers, and more extensively, we showcase the state-of-the-art in biocatalytic technologies that are enabling synthesis of diverse upcycled monomeric starting materials for a wide variety of macromolecules. Overall, this review emphasizes the importance of functional group interconversion as a promising strategy by which biocatalysis can aid the diversification and upcycling of monomers.

Fabrication of Mesoporous Materials Based on Supramolecular Self-Assembly of Guanosine Monophosphonate-Nickel Chloride (GMP-Ni) for High-Performance Hybrid Supercapacitors.

Supramolecular self-assembly of nickel chloride and guanosine mono-phosphonate (GMP) and nickel (Ni)-based GMP-Ni and their calcinated mesoporous electrode materials GMP-Ni-500 and GMP-Ni-700 at 500 and 700 °C, respectively, have been fabricated. GMP-Ni, GMP-Ni-500, and GMP-Ni-700 are examined for their supercapacitor performance in a three-electrode configuration. The electrochemical tests demonstrate the mesoporous battery-type nature of GMP-Ni-500 which exhibited a specific capacity (Cs) of about 289 C g-1 at 0.5 A g-1 current density. In addition, a cost-effective and simple asymmetric supercapacitor device has been fabricated with battery-type GMP-Ni-500 as a cathode material and capacitive-type activated carbon (AC) as an anodic material. In an operating voltage window of 0 to 1.5 V, hybrid supercapacitors (HSCs) based on GMP-Ni-500//AC exhibited a remarkable performance with a specific capacity (Cs) of 144 C g-1 at 0.5 A g-1. For the HSC device, the maximum of 66% capacity retention has been observed after 5000 charging/discharging cycles at 5 A g-1. Furthermore, the HSC device demonstrates a high energy density of 24 W h kg-1 at a power density of 297 W kg-1. The molecular transformation was established by employing theoretical calculations. These results suggest that our HSC has outstanding potential in technology development for next-generation commercial applications.

Olfactomedin-4 + neutrophils exacerbate intestinal epithelial damage and worsen host survival after Clostridioides difficile infection.

Neutrophils are key first responders to Clostridioides difficile infection (CDI). Excessive tissue and blood neutrophils are associated with worse histopathology and adverse outcomes, however their functional role during CDI remains poorly defined. Utilizing intestinal epithelial cell (IEC)-neutrophil co-cultures and a pre-clinical animal model of CDI, we show that neutrophils exacerbate C. difficile -induced IEC injury. We utilized cutting-edge single-cell transcriptomics to illuminate neutrophil subtypes and biological pathways that could exacerbate CDI-associated IEC damage. As such, we have established the first transcriptomics atlas of bone marrow (BM), blood, and colonic neutrophils after CDI. We found that CDI altered the developmental trajectory of BM and blood neutrophils towards populations that exhibit gene signatures associated with pro-inflammatory responses and neutrophil-mediated tissue damage. Similarly, the transcriptomic signature of colonic neutrophils was consistent with hyper-inflammatory and highly differentiated cells that had amplified expression of cytokine-mediated signaling and degranulation priming genes. One of the top 10 variable features in colonic neutrophils was the gene for neutrophil glycoprotein, Olfactomedin 4 (OLFM4). CDI enhanced OLFM4 mRNA and protein expression in neutrophils, and OLFM4 + cells aggregated to areas of severe IEC damage. Compared to uninfected controls, both humans and mice with CDI had higher concentrations of circulating OLFM4; and in mice, OLFM4 deficiency resulted in faster recovery and better survival after infection. Collectively, these studies provide novel insights into neutrophil-mediated pathology after CDI and highlight the pathogenic role of OLFM4 + neutrophils in regulating CDI-induced IEC damage. One Sentence Summary: Utilizing single-cell transcriptomics, IEC-epithelial co-cultures, and pre-clinical models of CDI, we have identified a subset of neutrophils that are marked by OLFM4 expression as pathogenic determinants of IEC barrier damage after CDI.

Evaluation of System-Level, Passive Chlorination in Gravity-Fed Piped Water Systems in Rural Nepal.

Over 2 billion people globally lack access to safely managed drinking water. In contrast to the household-level, manually implemented treatment products that have been the dominant strategy for gaining low-cost access to safe drinking water, passive chlorination technologies have the potential to treat water and reduce reliance on individual behavior change. However, few studies exist that evaluate the performance and costs of these technologies over time, especially in small, rural systems. We conducted a nonrandomized evaluation of two passive chlorination technologies for system-level water treatment in six gravity-fed, piped water systems in small communities in the hilly region of western Nepal. We monitored water quality indicators upstream of the treatment, at shared taps, and at households, as well as user acceptability and maintenance costs, over 1 year. At baseline, over 80% of tap samples were contaminated with Escherichia coli. After 1 year of system-level chlorination, only 7% of those same taps had E. coli. However, 29% of household stored water was positive for E. coli. Per cubic meter of treated water, the cost of chlorine was 0.06-0.09 USD, similar to the cost of monitoring technology installations. Safe storage, service delivery models, and reliable supply chains are required, but passive chlorination technologies have the potential to radically improve how rural households gain access to safely managed water.

Exosomal PTEN as a Predictive Marker of Aggressive Gliomas.

Background: Liquid biopsies have emerged as convenient alternative diagnostic methods to invasive biopsies, by evaluating disease-specific biomarkers and monitoring the disease risk noninvasively. Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) is a potent tumor suppressor, and its deletion/mutations are common in gliomas. Objective: Evaluate the feasibility of non-invasive detection of PTEN and its downstream genes in serum exosomes of glioma patients. Materials and methods: PTEN, Yes-associated-protein 1 (YAP1), and lysyl oxidase (LOX) transcript expression were monitored through polymerase chain reaction (PCR) in serum exosomes and their paired tumor tissues. The impact of PTEN and its axis genes expression on the overall survival (OS) was monitored. Results: Out of the 106 glioma serum samples evaluated, PTEN was retained/lost in 65.4%/34.6% of the tumor samples while it was retained/lost in 67.1%/32.9% of their paired exosomal fractions. PTEN expression in both tissue and paired exosomal fractions was observed in 48.11% of the samples. Sanger sequencing detected three mutations (Chr10: 89720791(A>G), Chr10:89720749(C>T), and Chr10:89720850(A>G). Both PTEN-responsive downstream genes (YAP1) and LOX axis were upregulated in the PTEN-deficient samples. PTEN loss was associated with poor survival in the glioma patients (hazard ratio (HR) 0.68, confidence interval (CI): 0.35-1.31, P = 0.28). The OS of the exosomal PTEN cohort coincided with the tumor-tissue PTEN devoid group (HR 1.08, CI: 0.49-2.36, P = 0.85). While, old age yielded the worst prognosis; gender, location, and grade were not prognostic of OS in the multivariate analysis. Conclusions: PTEN and its responsive genes YAP1 and LOX can be detected in serum exosomes and can serve as essential tools for the non-invasive evaluation/identification of aggressive gliomas.

Aggregation induced emission (AIE) materials for mitochondria imaging.

Mitochondria are energy producing organelle of the eukaryotic cells. The main activities of mitochondria monitored by various marker molecules are autophagy detection, estimation of Reactive Oxygen Species (ROS), mitochondrial death and Photodynamic therapy in cancer cells. Due to the advantages of specificity and sensitivity, aggregation induced emission (AIE) is now popular for the mitochondria labeling. In this chapter, we would like to discuss three major types of AIEgens probe used in mitochondrial staining. There are three different types of AIEgens available for mitochondrial detection and sensing based on their different structural motifs. The first type of AIEgens is tetraphenylethene (TPE) based molecules. Due to simple engineering architecture, TPE based AIEgens are widely employed in bioimaging applications. AIEgen such as triphenylphosphine (TPP), and triphenylamine (TPA) are also employed as a novel building block. These are successfully used as exceptional lipid droplet (LD)-specific bio probes in cell imaging, assurance of cell combination, and photodynamic cancer cell removal. The third group is the miscellaneous AIEgens probe involved in mitochondria imaging.

Aggregation induced emission materials for tissue imaging.

Aggregation Induced Emission (AIE) has ample opportunities in the sensing and biomedical field. Recently, AIE molecular architecture, AIE nanoparticles (NPs) and AIE conjugated polymer (CP) probes have shown outstanding performance in bioimaging applications. In this chapter, we are summarizing the updated advancement in AIE based molecular, NP and CP probes for tissue imaging. We are focusing in vivo and in vitro tissue imaging with in-depth morphological and molecular information using microscopic techniques. Also, we have summarized various infrared windows for fluorescence microscopic technique to achieve deep penetration and high resolution of tissue images. In addition, future difficulties and challenges of AIEgens in tissue imaging are shortly discussed.

Embryonal tumors with multilayered rosettes: A tertiary care centre experience.

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) is an extremely rare and highly aggressive tumor. It includes three distinct entities i.e, embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma (EBL) and medulloepithelioma (MEPL). Here, we present our institutional experience of seven ETMR cases treated over a period of five years. MATERIALS AND METHODS: Patients' records from 2015 to 2019 were reviewed manually and electronically to retrieve the data. Clinicopathological and outcome details of ETMR cases were entered in a predesigned proforma. RESULTS: A total of seven cases of ETMR were registered from 2015 to 2019 with a median age at presentation of four years (range 3-7 years). All patients underwent surgery. However, only three patients completed the planned adjuvant treatment, comprising of focal radiotherapy (RT) alone, craniospinal irradiation (CSI) alone and CSI followed by six cycles of chemotherapy in one patient each respectively. Two patients commenced CSI but deteriorated during RT and thereafter needed best supportive care. Two patients could not be started on any adjuvant treatment. Unfortunately, six patients succumbed to their disease within one year of their diagnosis. Only one patient who received both CSI and adjuvant chemotherapy is alive at 15 months of diagnosis. CONCLUSION: ETMR is a rare and aggressive entity. Majority of the patients die within one year of the diagnosis despite multimodality treatment.

Effect of prophylactic granulocyte-colony stimulating factor (G-CSF) on acute hematological toxicity in medulloblastoma patients during craniospinal irradiation (CSI).

OBJECTIVES: Haematological toxicity and treatment breaks are common during cranio-spinal irradiation (CSI) due to irradiation of large volume of bone marrow. We conducted this study to see the effect of prophylactic granulocyte colony stimulating factor (GCSF) in reducing treatment breaks. PATIENTS AND METHODS: The study was conducted over a period of 15 months from August 2017 to November 2018. Histopathologically proven Medulloblastoma patients received prophylactic GCSF during CSI. Acute hematological toxicities and treatment breaks were noted and effect of age and pretreatment blood counts were analyzed by SPSS (Statistical Package for Social Sciences) version 23. RESULTS: A total of 28 patients were included in the study. During CSI, hematological toxicity leading to treatment breaks was observed in 11 (39.3 %) patients, of which grade 3 and 2 toxicities were seen in ten and one patients respectively. Younger age (<10 years) at diagnosis was significantly associated with the development of hematological toxicity (p = 0.028, Chi-Square). No correlation was found with pre-treatment blood counts. CONCLUSION: Prophylactic use of GCSF may be effective in preventing radiation induced hematological toxicity and treatment breaks.

Effectiveness of intravenous immunoglobulin use in heparin-induced thrombocytopenia.

: Heparin-induced thrombocytopenia (HIT) syndrome is an immune-mediated disorder producing thrombocytopenia and thrombosis, with or without prior exposure to heparin. Although avoidance of heparin products and nonheparin anticoagulants are used, immune-based therapies including intravenous immunoglobulin (IVIg) have been tried when the thrombocytopenia persists or there is breakthrough thrombosis. We sought to systematically review and analyze the published literature on use of IVIg in the treatment of HIT. A systematic search of PubMed, Google Scholar, EMBASE and SCOPUS for all study designs and reports were carried out from inception until April 2019. Statistical analysis was done using Microsoft Excel and Stata version 13. In 34 patients with HIT, the mean age was 60 years. About 70% cases were by unfractionated heparin exposure and 30% by low-molecular weight heparin. The most common precipitant in the patients without heparin exposure was recent surgery. Average nadir platelet count for which IVIg was used was 28 000/µl. Time from resolution of the thrombocytopenia after IVIg treatment was 3 days with average platelet count recovery to 159 000/µl. Mean time from diagnosis to administration of IVIg was day 18. Thrombosis was identified in 32% of patients. About 77% patients improved (platelet count >100 000/µl or cessation of thrombosis) following use of IVIg. Logistic regression did not identify any factors that predicted IVIg response (P > 0.05). No thrombotic events or other adverse events were noted with use of IVIg. IVIg appears to be a safe and effective treatment option for HIT-related thrombocytopenia and for refractory thrombosis.

Comparative evaluation of efficacy and latency of twin mix vs 2% lignocaine HCL with 1:80000 epinephrine in surgical removal of impacted mandibular third molar.

INTRODUCTION: A prospective randomized double-blind controlled trial was conducted to evaluate the latency and duration of pterygomandibular nerve block with a mixture of 1.8 ml 2% lignocaine with 1:80,000 epinephrine and 1 ml of 4 mg dexamethasone and its impact on postoperative sequelae after surgical extraction of impacted mandibular third molars. MATERIALS AND METHODS: This study was conducted in 40 subjects referred to the department of oral and maxillofacial surgery; they were divided into 20 subjects each in group A and B with the age range of 18-72 years planned for elective surgical removal of unilateral impacted mandibular third molar. Each patient was randomly selected to receive anesthesia using 1.8 ml 2% lignocaine with 1:80,000 epinephrine in group A or 2.8 ml twin mix (1.8 ml 2% lignocaine with 1:80,000 epinephrine + 1 ml 4 mg dexamethasone) in group B. After injection of the anesthetic solution, the time to anesthetic effect, duration of anesthesia from initial patient perception of the anesthetic effect to the time when the effect subsides, need to reanesthetize the surgical site were recorded, and 10-point visual analog scale (VAS) was used to subjectively assess the overall pain intensity while injecting the study drug, during surgery, and in the postoperative period. RESULTS: Mean VAS value for pain on local anesthetic injection was less in twin-mix group. The time of onset of the local anesthetic was significantly less for the study group T, 51.35 ± 7.15 s when compared with patients in study group C (P less than 0.0001). The duration of soft tissue anesthesia was longer for all the patients in the study group T. On comparative evaluation between study group C and study group T, patients in the control group had more severe swelling and reduction in mouth opening in the postoperative period. CONCLUSION: The addition of dexamethasone to lignocaine and its administration as an intraspace injection significantly shortens the latency and prolongs the duration of the soft tissue anesthesia, with improved quality of life in the postoperative period after surgical extraction of mandibular third molars.

Deep androgen receptor suppression in prostate cancer exploits sexually dimorphic renal expression for systemic glucocorticoid exposure.

BACKGROUND: Enzalutamide and apalutamide are potent next-generation androgen receptor (AR) antagonists used in metastatic and non-metastatic prostate cancer. Metabolic, hormonal and immunologic effects of deep AR suppression are unknown. We hypothesized that enzalutamide and apalutamide suppress 11ß-hydroxysteroid dehydrogenase-2 (11ß-HSD2), which normally converts cortisol to cortisone, leading to elevated cortisol concentrations, increased ratio of active to inactive glucocorticoids and possibly suboptimal response to immunotherapy. On-treatment glucocorticoid changes might serve as an indicator of active glucocorticoid exposure and resultant adverse consequences. PATIENTS AND METHODS: Human kidney tissues were stained for AR and 11ß-HSD2 expression. Patients in three trials [neoadjuvant apalutamide plus leuprolide, enzalutamide ± PROSTVAC (recombinant poxvirus prostate-specific antigen vaccine) for metastatic castration-resistant prostate cancer (CRPC) and enzalutamide ± PROSTVAC for non-metastatic castration-sensitive prostate cancer] were analyzed for cortisol and its metabolites using liquid chromatography-mass spectrometry (LC-MS/MS). Progression-free survival was determined in the metastatic CRPC study of enzalutamide ± PROSTVAC for those with glucocorticoid changes above and below the median. RESULTS: Concurrent AR and 11ß-HSD2 expression occurs only in the kidneys of men. A statistically significant rise in cortisol concentration, cortisol/cortisone ratio and tetrahydrocortisol/tetrahydrocortisone ratio with AR antagonist treatment occurred uniformly across all three trials. In the trial of enzalutamide ± PROSTVAC for metastatic CRPC, high cortisol/cortisone ratio in the enzalutamide arm was associated with significantly improved progression-free survival. However, in the enzalutamide + PROSTVAC arm, the opposite trend was observed. CONCLUSION: Enzalutamide and apalutamide treatment toggles renal 11ß-HSD2 and significantly increases indicators of and exposure to biologically active glucocorticoids, which is associated with clinical outcomes.

Clinical and Health Care Utilization Outcomes of Kidney Transplantation in HIV-Positive Recipients: A Nationwide Analysis From 2008-2013.

BACKGROUND: Kidney transplantation (KTxp) provides dialysis independence, improved quality of life, and prolonged life expectancy for patients with end-stage renal disease. Before and during the early antiretroviral therapy era, KTxp was not a consideration for patients with end-stage renal disease who were HIV-positive (HIV+ve) given their short life expectancy and prevailing organ procurement constraints. Recent advancements in antiretroviral therapy and HIV care have overhauled this paradigm and KTxp has now been performed for several years, however, pragmatic studies documenting outcomes are lacking. We herein document the effectiveness of KTxp in patients who are HIV+ve by reporting clinical and health care utilization outcomes in the United States. METHODS: Utilizing the Inpatient Databases of the Healthcare Cost & Utilization Project spanning 2008-2013, we identified all adult recipients of KTxp by procedural codes and recipients who are HIV+ve by ICD-9 codes. We extracted demographic, clinical, and resource utilization variables and compared recipients who are HIV+ve with those who are HIV-negative (HIV-ve). We then performed descriptive statistics and multivariate analysis using logistic regression to assess the effect of HIV on clinical and utilization outcomes. RESULTS: A total of 104,137 patients had kidney transplants during the study period. Of the total, 605 patients were HIV+ve. Infections rates were similar among patients who were HIV+ve and HIV-ve (odds ratio [OR] 1.18, confidence interval [CI] 0.58-2.40; P = .652). In-hospital mortality rates were also similar (OR 0.83, CI 0.18-3.69; P = .80). Hospital charges for patients who were HIV+ve were no different from patients who were HIV-ve ($195,099 ± 1074 vs $186,567 ± 9558; P = .38). CONCLUSION: Clinical and fiscal outcomes are comparable among patients who are HIV+ve and HIV-ve during transplant hospitalization.

T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts.

Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter. To improve targeting efficiency, guide RNA design was found to be tolerant to three mismatches or up to three additional bases appended to the 5' end. The addition of three guanines to a T7-based guide RNA improved guide RNA expression 80-fold and achieved transcriptional output similar to the strong Pol III snr52 promoter. Resulting gene editing and dCas9-guided gene regulation with a T7-based guide RNA was on par with the commonly used snr52 system in S. cerevisiae. Finally, 96% and 60% genome editing efficiencies were achieved in Kluyveromyces lactis and Yarrowia lipolytica respectively with minimal optimization of this system. Thus, T7-based expression of sgRNAs offers an orthogonal method for implementing CRISPR systems in fungal systems.

Leptin receptor q223r polymorphism influences neutrophil mobilization after Clostridium difficile infection.

Clostridium difficile is the leading cause of nosocomial infections in the United States. Clinical disease outcomes after C. difficile infection (CDI) are dependent on intensity of host inflammatory responses. Specifically, peak peripheral white blood cell (WBC) count >20 × 109 l-1 is an indicator of adverse outcomes in CDI patients, and is associated with higher 30-day mortality. We show that homozygosity for a common single nucleotide polymorphism (Q to R mutation in leptin receptor that is present in up to 50% of people), significantly increases the risk of having peak peripheral WBC count >20 × 109 l-1 (odds ratio=5.41; P=0.0023) in CDI patients. In a murine model of CDI, we demonstrate that mice homozygous for the same single nucleotide polymorphism (RR mice) have more blood and tissue leukocytes (specifically neutrophils), exaggerated tissue inflammation, and higher mortality as compared with control mice, despite similar pathogen burden. Further, we show that neutrophilia in RR mice is mediated by gut microbiota-directed expression of CXC chemokine receptor 2 (CXCR2), which promotes the release of neutrophils from bone marrow reservoir. Overall these studies provide novel mechanistic insights into the role of human genetic polymorphisms and gut microbiota in regulating the fundamental biological process of CDI-induced neutrophilia.

Yeast Terminator Function Can Be Modulated and Designed on the Basis of Predictions of Nucleosome Occupancy.

The design of improved synthetic parts is a major goal of synthetic biology. Mechanistically, nucleosome occupancy in the 3' terminator region of a gene has been found to correlate with transcriptional expression. Here, we seek to establish a predictive relationship between terminator function and predicted nucleosome positioning to design synthetic terminators in the yeast Saccharomyces cerevisiae. In doing so, terminators improved net protein output from these expression cassettes nearly 4-fold over their original sequence with observed increases in termination efficiency to 96%. The resulting terminators were indeed depleted of nucleosomes on the basis of mapping experiments. This approach was successfully applied to synthetic, de novo, and native terminators. The mode of action of these modifications was mainly through increased termination efficiency, rather than half-life increases, perhaps suggesting a role in improved mRNA maturation. Collectively, these results suggest that predicted nucleosome depletion can be used as a heuristic approach for improving terminator function, though the underlying mechanism remains to be shown.

Effect of Statins on the Mortality of Bacteremic Patients: A Systematic Review and Meta-analysis of Clinical Trials.

BACKGROUND: Statins modify inflammatory cell signaling during the immune response to infection. This has been considered as a pleotropic effect. Effects of statins in inflammatory conditions such as bacteremia have been found to be controversial. AIMS: We examined the effect of statins on the mortality of bacteremia patients. MATERIALS AND METHODS: Major databases were searched for the pertinent clinical trials. RESULTS: Six cohort studies comprising 7553 patients were included. Hospital mortality was lower (15.36% vs 22.28%) in patients on statin. CONCLUSIONS: There may be a potential role of statins in similar inflammatory and infective conditions.