Convergent genetic adaptation of Escherichia coli in minimal media leads to pleiotropic divergence.

Adaptation in an environment can either be beneficial, neutral or disadvantageous in another. To test the genetic basis of pleiotropic behaviour, we evolved six lines of E. coli independently in environments where glucose and galactose were the sole carbon sources, for 300 generations. All six lines in each environment exhibit convergent adaptation in the environment in which they were evolved. However, pleiotropic behaviour was observed in several environmental contexts, including other carbon environments. Genome sequencing reveals that mutations in global regulators rpoB and rpoC cause this pleiotropy. We report three new alleles of the rpoB gene, and one new allele of the rpoC gene. The novel rpoB alleles confer resistance to Rifampicin, and alter motility. Our results show how single nucleotide changes in the process of adaptation in minimal media can lead to wide-scale pleiotropy, resulting in changes in traits that are not under direct selection.

Cell cycle regulators and bone: development and regeneration.

Cell cycle regulators act as inhibitors or activators to prevent cancerogenesis. It has also been established that they can play an active role in differentiation, apoptosis, senescence, and other cell processes. Emerging evidence has demonstrated a role for cell cycle regulators in bone healing/development cascade. We demonstrated that deletion of p21, a cell cycle regulator acting at the G1/S transition enhanced bone repair capacity after a burr-hole injury in the proximal tibia of mice. Similarly, another study has shown that inhibition of p27 can increase bone mineral density and bone formation. Here, we provide a concise review of cell cycle regulators that influence cells like osteoblasts, osteoclasts, and chondrocytes, during development and/or healing of bone. It is imperative to understand the regulatory processes that govern cell cycle during bone healing and development as this will pave the way to develop novel therapies to improve bone healing after injury in instances of aged or osteoporotic fractures.

Determination of albuterol and montelukast post-expiry drug strength by HPLC.

Background: Knowing the level of active ingredients in an expired drug is a matter of concern irrespective of its final disposition. This is also a matter of national security and defense as it has important implications on the nation's stockpile of prescription medications. Current literature has limited information about the strength of expired medications and any relevant trends. Objective: To utilize high performance liquid chromatography (HPLC) to determine the strength of selected drugs for asthma and chronic obstructive pulmonary disease (COPD) as a class of therapeutic agents commonly used in free clinics. Methods: Samples from expired lots of montelukast and albuterol pharmaceutical products were analyzed for their levels of their respective active ingredients. Two HPLC methods were developed, validated, and applied to achieve this goal. Quantitative analysis of each drug was performed using two different reversed phase C18 columns with a linear gradient of acetonitrile in 0.1% aqueous formic acid at a flow rate of 1 mL/min for both methods. Detection wavelength for montelukast and albuterol was 280 and 277 nm, respectively. Results: Expiry dates of analyzed batches ranged from 2003 to 2019. Despite the extended time range beyond expiry dates, levels of both drugs were relatively consistent and exceeded 90% of the listed strength in most analyzed lots. Conclusions: Our results introduce a new perspective towards reducing the financial burden resulting from disposal of expired medications with retained strength. They also offer supporting evidence to extend the use of out-of-date montelukast and albuterol preparations at home and in free clinics.

Astrocytes influence medulloblastoma phenotypes and CD133 surface expression.

The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.

Autosomal dominant tubulointerstitial kidney disease-UMOD is the most frequent non polycystic genetic kidney disease.

BACKGROUND: Autosomal dominant tubulointerstitial kidney disease (ADTKD) caused by mutations in the UMOD gene (ADTKD-UMOD) is considered rare and often remains unrecognised. We aimed to establish the prevalence of genetic kidney diseases, ADTKD and ADTKD-UMOD in adult chronic kidney disease (CKD) patients, and to investigate characteristic features. METHODS: We sent questionnaires on family history to all patients with CKD stages 3-5 in our tertiary renal centre to identify patients with inherited renal disease. Details on clinical and family history were obtained from patient interviews and clinical records. Sanger sequencing of the UMOD gene was performed from blood or saliva samples. RESULTS: 2027 of 3770 sent questionnaires were returned. 459 patients reported a family history, which was consistent with inherited kidney disease in 217 patients. 182 non-responders with inherited kidney diseases were identified through a database search. Of these 399 individuals, 252 had autosomal dominant polycystic kidney disease (ADPKD), 28 had ADTKD, 25 had Alports, and 44 were unknown, resulting in 11% of CKD 3-5 patients and 19% of end-stage renal disease patients with genetic kidney diseases. Of the unknown, 40 were genotyped, of whom 31 had findings consistent with ADTKD. 30% of unknowns and 39% of unknowns with ADTKD had UMOD mutations. Altogether, 35 individuals from 18 families were found to have ten distinct UMOD mutations (three novel), making up 1% of patients with CKD 3-5, 2% of patients with end-stage renal disease, 9% of inherited kidney diseases and 56% with ADTKD. ADTKD-UMOD was the most common genetic kidney disease after ADPKD with a population prevalence of 9 per million. Less proteinuria and haematuria, but not hyperuricaemia or gout were predictive of ADTKD-UMOD. The main limitations of the study are the single-centre design and a predominantly Caucasian population. CONCLUSIONS: The prevalence of genetic kidney diseases and ADTKD-UMOD is significantly higher than previously described. Clinical features poorly predicted ADTKD-UMOD, highlighting the need for genetic testing guided by family history alone.

Dual Specificity Phosphatase 5-Substrate Interaction: A Mechanistic Perspective.

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members' abilities to remove phosphate groups from serine/threonine and tyrosine residues. We provide a comparison of DUSP5's structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5's mechanistic interaction and specificity toward phospho-extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen-activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449-1461, 2017.

Dual Specificity Phosphatase 5 Is Essential for T Cell Survival.

The mitogen-activated protein kinase (MAPK) pathway regulates many key cellular processes such as differentiation, apoptosis, and survival. The final proteins in this pathway, ERK1/2, are regulated by dual specificity phosphatase 5 (DUSP5). DUSP5 is a nuclear, inducible phosphatase with high affinity and fidelity for ERK1/2. By regulating the final step in the MAPK signaling cascade, DUSP5 exerts strong regulatory control over a central cellular pathway. Like other DUSPs, DUSP5 plays an important role in immune function. In this study, we have utilized new knockout mouse reagents to explore its function further. We demonstrate that global loss of DUSP5 does not result in any gross phenotypic changes. However, loss of DUSP5 affects memory/effector CD8+ T cell populations in response to acute viral infection. Specifically, Dusp5-/- mice have decreased proportions of short-lived effector cells (SLECs) and increased proportions of memory precursor effector cells (MPECs) in response to infection. Further, we show that this phenotype is T cell intrinsic; a bone marrow chimera model restricting loss of DUSP5 to the CD8+ T cell compartment displays a similar phenotype. Dusp5-/- T cells also display increased proliferation, increased apoptosis, and altered metabolic profiles, suggesting that DUSP5 is a pro-survival protein in T cells.

Prevalence of serum anti M-type phospholipase A2 receptor antibody in primary membranous nephropathy: A single center experience.

We conducted a prospective study to assess utility of detection of antibodies to phospholipase A2receptor (PLA2R) in the serum of patients with membranous nephropathy. Seventy five patients with biopsy proven membranous nephropathy admitted between January 2011 and September 2014 were studied. Serum anti- PLA2R was tested by indirect immunofluorescence. The test was positive in 45 out of 60 patients with primary membranous nephropathy (PMN) and in none of the 15 patients with secondary membranous nephropathy, with a sensitivity of 75% and specificity of 100% for PMN. Anti PLA2R positivity also showed a significant correlation with quantum of proteinuria and negative correlation with serum albumin. This study has validated detection of serum anti PLA2R in PMN as a non invasive diagnostic tool in Indian patients.

Isolated Complete Jejunal Transection After Blunt Abdominal Trauma: CT Imaging.

Abdominal injury following road traffic accident is less common, compared to the extremities, head and chest. Bowel may get injured following blunt abdominal trauma, but perforation and complete transection is rare. Initial clinical examination may be unreliable, as signs of bowel injury may take some time to develop. Imaging plays a crucial role in the early and accurate diagnosis of bowel injuries. We report a case of 21-year-old male, who presented with severe abdominal pain, following a road traffic accident. Chest X-Ray was normal and abdominal ultrasound revealed intra-peritoneal free fluid with internal echoes. Contrast enhanced CT scan showed pneumoperitoneum and intraperitoneal free fluid with disruption in continuity of proximal jejunum along with signs of shock bowel and bowel ischemia. This report highlights the role of CT imaging in the prompt diagnosis of bowel transection following blunt abdominal trauma.

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets.

BACKGROUND: Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5. METHODS: pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate. RESULTS: The most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 µM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD (3) _2320) with IC50 of 33 µM that is similar to NTS and does not aggregate. CONCLUSIONS: The new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs.

Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins.

BACKGROUND: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. RESULTS: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. CONCLUSION: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

Connexin modulators and their potential targets under the magnifying glass.

Gap junction channels, assembled from connexins, mediate communication and signaling between adjacent cells by allowing the passage of ions, metabolites and signaling molecules. Their physiological significance and importance in cellular homeostasis is reflected by the fact that their dysfunction leads to a multitude of pathologies. However, the complex physiological and pathophysiological roles of connexins are not well understood. Therefore, pharmacological tools to further elucidate their functions and to validate them as drug targets for the development of novel therapies for connexin-based diseases are urgently needed. In this article we will review diseases caused by mutations, abnormal expression and function of connexins (e.g. deafness, atrial fibrillation and other cardiac arrhythmias, peripheral and central nervous system neuropathies, epilepsy, and cancer) and we will discuss the role of connexins as potential therapeutic targets. This will be followed by a detailed overview of the different classes of modulators including proposed mechanisms of action, selectivity and structure-activity relationships. Classical connexin channel uncoupling agent like long-chain alcohols (e.g. heptanol), glycyrrhetinic acid and its derivatives, later discovered connexin blocking chemotypes like 2-aminophenoxyborates, fenamates, quinines and triphenylmethanes, as well as gap junctional coupling enhancing compounds like antiarrhythmic peptides will be discussed.

The cancer stem cell paradigm: a new understanding of tumor development and treatment.

IMPORTANCE OF THE FIELD: Cancer is the second leading cause of death in the United States, and therefore remains a central focus of modern medical research. Accumulating evidence supports a 'cancer stem cell' (CSC) model - where cancer growth and/or recurrence is driven by a small subset of tumor cells that exhibit properties similar to stem cells. This model may provide a conceptual framework for developing more effective cancer therapies that target cells propelling cancer growth. AREAS COVERED IN THIS REVIEW: We review evidence supporting the CSC model and associated implications for understanding cancer biology and developing novel therapeutic strategies. Current controversies and unanswered questions of the CSC model are also discussed. WHAT THE READER WILL GAIN: This review aims to describe how the CSC model is key to developing novel treatments and discusses associated shortcomings and unanswered questions. TAKE HOME MESSAGE: A fresh look at cancer biology and treatment is needed for many incurable cancers to improve clinical prognosis for patients. The CSC model posits a hierarchy in cancer where only a subset of cells drive malignancy, and if features of this model are correct, has implications for development of novel and hopefully more successful approaches to cancer therapy.

KDIGO clinical practice guideline for the care of kidney transplant recipients: a summary

The 2009 Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guideline on the monitoring, management, and treatment of kidney transplant recipients is intended to assist the practitioner caring for adults and children after kidney transplantation. The guideline development process followed an evidence-based approach, and management recommendations are based on systematic reviews of relevant treatment trials. Critical appraisal of the quality of the evidence and the strength of recommendations followed the Grades of Recommendation Assessment, Development, and Evaluation (GRADE) approach. The guideline makes recommendations for immunosuppression and graft monitoring, as well as prevention and treatment of infection, cardiovascular disease, malignancy, and other complications that are common in kidney transplant recipients, including hematological and bone disorders. Limitations of the evidence, especially the lack of definitive clinical outcome trials, are discussed and suggestions are provided for future research. This summary includes a brief description of methodology and the complete guideline recommendations but does not include the rationale and references for each recommendation, which are published elsewhere.

An unusual non-immunological cause of renal pulmonary syndrome.

A 38-year-old Caucasian male presented with a 4-week history of nose bleeds, gross hematuria and blurred vision. He was a smoker, who had used cannabis and cocaine previously. At presentation, he had features of malignant hypertension (blood pressure 220/120 mmHg), was hypoxic on room air, with no signs of fluid overload or heart failure. He had acute renal failure with radiological evidence of alveolar hemorrhage. Renal biopsy showed extensive ischemic collapse of glomeruli and severe fibrointimal thickening of the arteries with fibrinoid deposits in the wall. Auto-immune screen was negative. Serum creatinine peaked at 749 micromol/l. Adequate control of blood pressure and supportive oxygen therapy lead to a complete clinical and radiological resolution of the pulmonary hemorrhage and he did not need dialysis. Eighteen months on, his serum creatinine is stable at 279 micromol/l with good blood pressure control. Malignant hypertension is not a recognized cause of the renal-pulmonary syndrome and physicians should be aware of the possibility, if only to avoid inappropriate treatments like plasmapheresis and immunosuppression. History of cocaine use is important in the setting of an acute vascular event.

APOE gene polymorphisms and response to statin therapy.

Published studies investigating the role of APOE gene on lipid response (total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides) to statin treatment have reported inconsistent results. A meta-analysis was conducted to estimate the lipid response to statin treatment among APOE genetic variants (e2 carriers, e3e3 homozygotes and e4 carriers). Twenty-four studies were included in the meta-analyses. The pooled mean reduction (Delta mu) in TC from baseline was significant for all variants (e2 carriers: Delta mu=-27.7% (-32.5 to -22.8%), e3e3: Delta mu=-25.3% (-28.0 to -22.6%) and e4 carriers: Delta mu=-25.1% (-29.3 to -21.0%)). Significant changes in LDL-C, HDL-C and triglyceride levels were also noted for all genotypes, although these changes did not differ significantly among genotypic groups. There was significant heterogeneity among the studies. Given these non-significant effects of APOE genotypes on lipid responses, there is little reason to consider the use of APOE genetic testing for guiding treatment with statins.