Emycin-E purified from Streptomyces sp. RG1011 from Himalayan soil has antibiofilm activity against Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an opportunistic pathogen that causes deadly infections in human as well as animals. The intricate network of virulence factors and biofilms are the major hindrance for the antibiotics in the successful treatment of the infection. The aim of this study is to isolate, identify and characterize natural antimicrobial agent against S. aureus from natural resources. METHODS: Himalayan soils were subjected to primary, secondary and tertiary screening to isolate soil Actinobacteria. Identification and characterization of the isolate was done by various biochemical assays and 16s rDNA sequencing. Partial purification of the potent antimicrobial agent was done by n-butanol from the culture supernatant, TLC and HPLC were performed to purify the active component and subjected to FTIR and ESI-MS analysis. RESULTS: The potent isolate RM-1(13) was confirmed as Streptomyces griseus strain RG1011 (NCBI accession no: 0M780275) by biochemical and molecular analysis. The partially purified antimicrobial agent was active against various Gram-positive and Gram-negative pathogens. The active component was purified by HPLC and identified as Emycin-E by ESI-MS analysis. The Emycin-E has calculated MIC of 0.31 µg/ml against S. aureus ATCC 25923. Emycin-E inhibits the biofilm formation of S. aureus in in vitro microtiter plate assay. CONCLUSIONS: The identified antimicrobial agent was found active against various Gram-positive and Gram-negative pathogens. We have successfully identified the active compound as Emycin-E by FTIR and ESI-MS analysis. Our study suggests the role of Emycin-E in the inhibition of biofilm formation in S. aureus.

Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease.

The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.

Cellubrevin/vesicle-associated membrane protein-3-mediated endocytosis and trafficking regulate platelet functions.

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbß3, αvß3), and purinergic receptors (P2Y1, P2Y12), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble N-ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function. Although not required for normal platelet exocytosis or hemostasis, VAMP-3-/- mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Other granule markers were unaffected. Direct experiments, both in vitro and in vivo, showed that loss of VAMP-3 led to a robust defect in uptake/storage of Fg in platelets and cultured megakaryocytes. Uptake of the fluid-phase marker, dextran, was only modestly affected. Time-dependent uptake and endocytic trafficking of Fg and dextran were followed using 3-dimensional-structured illumination microscopy. Dextran uptake was rapid compared with Fg, but both cargoes progressed through Rab4+, Rab11+, and von Willebrand factor (VWF)+ compartments in wild-type platelets in a time-dependent manner. In VAMP-3-/- platelets, the 2 cargoes showed limited colocalization with Rab4, Rab11, or VWF. Loss of VAMP-3 also affected some acute platelet functions, causing enhanced spreading on Fg and fibronectin and faster clot retraction compared with wild-type. In addition, the rate of Janus kinase 2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3-/- platelets. Collectively, our studies show that platelets are capable of a range of endocytosis steps, with VAMP-3 being pivotal in these processes.

Preferential PPAR-α activation reduces neuroinflammation, and blocks neurodegeneration in vivo.

Neuroinflammation, immune reactivity and mitochondrial abnormalities are considered as causes and/or contributors to neuronal degeneration. Peroxisome proliferator-activated receptors (PPARs) regulate both inflammatory and multiple other pathways that are implicated in neurodegeneration. In the present study, we investigated the efficacy of fenofibrate (Tricor), a pan-PPAR agonist that activates PPAR-α as well as other PPARs. We administered fenofibrate to superoxide dismutase 1 (SOD1(G93A)) mice daily prior to any detectable phenotypes and then animal behavior, pathology and longevity were assessed. Treated animals showed a significant slowing of the progression of disease with weight loss attenuation, enhanced motor performance, delayed onset and survival extension. Histopathological analysis of the spinal cords showed that neuronal loss was significantly attenuated in fenofibrate-treated mice. Mitochondria were preserved as indicated by Cytochrome c immunostaining in the spinal cord, which maybe partly due to increased expression of the PPAR-γ co-activator 1-α. The total mRNA analysis revealed that neuroprotective and anti-inflammatory genes were elevated, while neuroinflammatory genes were down-regulated. This study demonstrates that the activation of PPAR-α action via fenofibrate leads to neuroprotection by both reducing neuroinflammation and protecting mitochondria, which leads to a significant increase in survival in SOD1(G93A) mice. Therefore, the development of therapeutic strategies to activate PPAR-α as well as other PPARs may lead to new therapeutic agents to slow or halt the progression of amyotrophic lateral sclerosis.

The cellular basis of platelet secretion: Emerging structure/function relationships.

Platelet activation has long been known to be accompanied by secretion from at least three types of compartments. These include dense granules, the major source of small molecules; α-granules, the major protein storage organelle; and lysosomes, the site of acid hydrolase storage. Despite ~60 years of research, there are still many unanswered questions about the cell biology of platelet secretion: for example, how are these secretory organelles organized to support cargo release and what are the key routes of cargo release, granule to plasma membrane or granule to canalicular system. Moreover, in recent years, increasing evidence points to the platelet being organized for secretion of the contents from other organelles, namely the dense tubular system (endoplasmic reticulum) and the Golgi apparatus. Conceivably, protein secretion is a widespread property of the platelet and its organelles. In this review, we concentrate on the cell biology of the α-granule and its structure/function relationships. We both review the literature and discuss the wide array of 3-dimensional, high-resolution structural approaches that have emerged in the last few years. These have begun to reveal new and unanticipated outcomes and some of these are discussed. We are hopeful that the next several years will bring rapid advances to this field that will resolve past controversies and be clinically relevant.

Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures.

Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown. Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte. In conclusion, we suggest that these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research.

Monitored Anesthesia Care (MAC) as an Alternative to General Anesthesia (GA): Prospective Double-Blinded Randomized Controlled Study Comparing Efficacy and Safety of Dexmedetomidine and Ketamine Infusions with Ultrasonography (USG) Guided Pectoral Nerve Block (PECs) for Postoperative Analgesia in Breast Surgery.

BACKGROUND AND PURPOSE: PECs blocks are usually combined for breast surgery under general anesthesia (GA) to provide postoperative analgesia rather than primary anesthesia technique. MATERIAL AND METHODS: A prospective, interventional, single-center, double-blind, randomized, parallel-group, active-controlled, Helsinki protocol-compliant clinical study was conducted in a tertiary care teaching center after obtaining the Ethics Committee's approval and patients' written informed consent. Forty-eight American Society of Anesthesiologists physical status I/II patients aged 18-60 years, undergoing elective unilateral breast surgery were enrolled. Patients were block-randomized (computer-generated) to two equal groups (24 patients each), with one of them receiving Dexmedetomidine and the other one Ketamine. For concealment, sequentially numbered, sealed, opaque envelopes were used. The study was double-blinded for both the anesthesiologist and outcome assessor anesthesiologist. Obese patients (body mass index > 30), those with infection at block site, coagulopathy and known hypersensitivity to local anesthetics or study medications as well as individuals who refused participation in research were all excluded. The Dexmedetomidine group received a bolus of 0.5 mcg/kg over ten minutes, followed by an infusion of 0.3 mcg/kg/hour, while the Ketamine group received a bolus of 0.5 mg/kg over ten minutes, followed by an infusion of 0.3 mg/kg/hour. Postoperative analgesia was compared using a visual analogue scale (VAS) at regular intervals. When VAS exceeded four, 1 mg/kg intravenous Pethidine was administered as a rescue analgesic. RESULTS: Sub-anesthetic low-dose Ketamine was more effective than low-dose Dexmedetomidine in prolonging PECs block analgesia, which was statistically significant (p value < 0.001). The Ketamine group had lower rescue analgesic Pethidine consumption and longer first-rescue analgesia demand time. There was no significant hemodynamic difference between the study groups. CONCLUSION: Ketamine was more efficient than Dexmedetomidine for postoperative analgesia.

Premature death of TDP-43 (A315T) transgenic mice due to gastrointestinal complications prior to development of full neurological symptoms of amyotrophic lateral sclerosis.

Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Transgenic mouse lines overexpressing wild-type or mutant TDP-43 exhibit ALS-like symptom, motor abnormalities and early paralysis followed by death. Reports on lifespan and phenotypic behaviour in Prp-TDP-43 (A315T) vary, and these animals are not fully characterized. Although it has been proposed that the approximate 20% loss of motor neurons at end stage is responsible for the severe weakness and death in TDP-43 mice, this degree of neurologic damage appears insufficient to cause death. Hence we studied these mice to further characterize and determine the reason for the death. Our characterization of TDP-43 transgenic mice showed that these mice develop ALS-like symptoms that later become compounded by gastrointestinal (GI) complications that resulted in death. This is the first report of a set of pathological evidence in the GI track that is strong indicator for the cause of death of Prp-hTDP-43 (A315T) transgenic mice.

Awareness of safety protocols for prevention of needle stick injuries in anaesthesiologists from Maharashtra: A survey study.

BACKGROUND AND AIM: Needle stick injury (NSI) has a serious risk of transmission of various blood borne pathogens amongst healthcare personnel and more so in anaesthesiologists. This survey assessed the prevalence of NSI and awareness of safety protocols for its prevention amongst the anaesthesiologists from Maharashtra, India. METHODS: This self-administered survey was completed by 403 anaesthesiologists across Maharashtra from August 2019 to October 2019. The pre-validated and pretested 18-item questionnaire was administered using Google forms and the link was circulated amongst anaesthesiologists electronically. The questionnaire items included information on the awareness of safety protocols and immediate measure after NSI, knowledge of immunisation and safety practices followed in routine practice. Data were collected, tabulated and coded in Microsoft Excel. Descriptives are presented for the different items and prevalence of NSI. Comparison of prevalence of NSI in subgroups based on gender, period of experience and type of practice were analysed using Chi-square test. RESULTS: The prevalence of NSI was 73.7% (n = 403) in anaesthesiologists with 71.1% (n = 235) in males and 77.4% (n = 168) in females. The anaesthesiologists from the medical schools had a prevalence of 75.0% (n = 148), those in private practice had a prevalence of 72.7% (n = 216), whereas those working in both medical school and private practice had a prevalence of 74.4% (n = 39). A greater prevalence was observed in those working for longer periods. CONCLUSION: The prevalence of NSI's is alarmingly high amongst anaesthesiologists and there is an immediate need of creating awareness and practice safety protocols in routine practice. Training and education are required in the formative years of healthcare curriculum.

3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

BACKGROUND: State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure. OBJECTIVES: Our goal was to determine the 3D organization of α-granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships. METHODS: We used serial block face-scanning electron microscopy images to render the 3D ultrastructure of α-granules, dense granules, mitochondria, canalicular system, and plasma membrane for 30 human platelets, 10 each from 3 donors. α-Granule compositional data were assessed by sequential, serial section cryo-immunogold electron microscopy and by immunofluorescence (structured illumination microscopy). RESULTS AND CONCLUSIONS: α-Granule number correlated linearly with platelet size, while dense granule and mitochondria number had little correlation with platelet size. For all subcellular compartments, individual organelle parameters varied considerably and organelle volume fraction had little correlation with platelet size. Three-dimensional data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of α-granules came within ≤35 nm of each other, a distance associated in other cell systems with protein-mediated contact sites. Size and shape analysis of the 1488 α-granules analyzed revealed no more variation than that expected for a Gaussian distribution. Protein distribution data indicated that all α-granules likely contained the same major set of proteins, albeit at varying amounts and varying distribution within the granule matrix.