On the Origin of Metallicity and Stability of the Metastable Phase in Chemically Exfoliated MoS2_2

Chemical exfoliation of MoS$_2$ via Li-intercalation route has led to many desirable properties and spectacular applications due to the presence of a metastable state in addition to the stable H phase. However, the nature of the specific metastable phase formed, and its basic charge conduction properties have remained controversial. Using spatially resolved Raman spectroscopy (~1 micrometer resolution) and photoelectron spectroscopy (~120 nm resolution), we probe such chemically exfoliated MoS$_2$ samples in comparison to a mechanically exfoliated H phase sample and confirm that the dominant metastable state formed by this approach is a distorted T' state with a small semiconducting gap. Investigating two such samples with different extents of Li residues present, we establish that Li+ ions, not only help to exfoliate MoS$_2$ into few layer samples, but also contribute to enhancing the relative stability of the metastable state as well as dope the system with electrons, giving rise to a lightly doped small bandgap system with the T' structure, responsible for its spectacular properties.Comment: 34 pages, Main manuscript + Supplementary Materia

Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD.Telethon-Italy and Autonomous Province of Trento (TCP12013 to M.P.); Association Française contre les Myopathies (AFM-22221 to M.P. and M.B.); PRIN-MUR (2017F2A2C5 to M.P.); National Institutes of Health (1R21NS111768-01 to M.P. and U.B.P.); PROGRAM RARE DISEASES CNCCS-Scarl-Pomezia (M.P.); FONDAZIONE AIRC-Italy (24423 to M.P.); Alzheimer Trento Onlus with the legato Baldrachi (M.B.); the Agence Nationale de la Recherche (ANR-15-JPWG-0003-05 JPND CIRCPROT and ANR-18-CE16-0009-01 AXYON to F.S.) and the Spanish Ministry of Science, Innovation and Universities (RTI2018-096322-B-I00 MCIU/AEI/FEDER-UE to J.J.L.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields

Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize

A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells

Betulinic acid (BA), a member of pentacyclic triterpenes has shown important biological activities like anti-bacterial, anti-malarial, anti-inflammatory and most interestingly anticancer property. To overcome its poor aqueous solubility and low bioavailability, structural modifications of its functional groups are made to generate novel lead(s) having better efficacy and less toxicity than the parent compound. BA analogue, 2c was found most potent inhibitor of colon cancer cell line, HT-29 cells with IC50 value 14.9 μM which is significantly lower than standard drug 5-fluorouracil as well as parent compound, Betulinic acid. We have studied another mode of PCD, autophagy which is one of the important constituent of cellular catabolic system as well as we also studied proteasomal degradation pathway to investigate whole catabolic pathway after exploration of 2c on HT-29 cells. Mechanism of autophagic cell death was studied using fluorescent dye like acridine orange (AO) and monodansylcadaverin (MDC) staining by using fluorescence microscopy. Various autophagic protein expression levels were determined by Western Blotting, qRT-PCR and Immunostaining. Confocal Laser Scanning Microscopy (CLSM) was used to study the colocalization of various autophagic proteins. These were accompanied by formation of autophagic vacuoles as revealed by FACS and transmission electron microscopy (TEM). Proteasomal degradation pathway was studied by proteasome-Glo™ assay systems using luminometer.The formation of autophagic vacuoles in HT-29 cells after 2c treatment was determined by fluorescence staining – confirming the occurrence of autophagy. In addition, 2c was found to alter expression levels of different autophagic proteins like Beclin-1, Atg 5, Atg 7, Atg 5-Atg 12, LC3B and autophagic adapter protein, p62. Furthermore we found the formation of autophagolysosome by colocalization of LAMP-1 with LC3B, LC3B with Lysosome, p62 with lysosome. Finally, as proteasomal degradation pathway downregulated after 2c treatment colocalization of ubiquitin with lysosome and LC3B with p62 was studied to confirm that protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway. In summary, betulinic acid analogue, 2c was able to induce autophagy in HT-29 cells and as proteasomal degradation pathway downregulated after 2c treatment so protein degradation in autophagy induced HT-29 cell

Optimization and kinetics studies on biodegradation of atrazine using mixed microorganisms

In this work, degradation of atrazine was carried out in batch reactors using mixed microorganisms obtained from pharmaceutical wastewater sludge. The effects of process parameters like pH, temperature, inoculum concentration, and agitation speed on atrazine degradation were studied and optimized using response surface methodology (RSM). The optimum condition for the maximum degradation of atrazine was pH – 6.7, temperature – 29.3 °C, inoculum concentration – 5%, and agitation speed – 137 rpm. At these conditions, the effect of atrazine concentration was studied. From the results, it was found that increase in atrazine concentration decreases the degradation efficiency. The maximum atrazine degradation was found to be 94.4%. Various cell growth models and substrate inhibition models were used to describe the atrazine degradation kinetics. From the results, it was found that Haldane model fits the data well with R2 value of 0.9001

Detection of metallo β-lactamase producing Klebsiella pneumoniae in a neonatal septicemia

A 10-day old preterm neonate, appropriate for the date, was admitted for lethargy and feeding intolerance. By culturing its blood, the antibiogram of the causative bacterium was ascertained by both Kirby-Bauer disc diffusion method and the Vitek2 system. Due clinical steps were taken for survival of the baby. The baby was suffering from septicemia. The causative bacterium was Klebsiella pneumoniae (K. pneumoniae). The isolated strain was found resistant to a total of 17 antibiotics; the strain was positive for extended spectrum β-lactamase production and resistant to two carbapenems, imipenem and meropenem. The baby could not survive. The baby was infected with an appalling strain of K. pneumoniae with a capacity to produce metallo β-lactamase overriding carbapenems, which is found to present in the state of Odisha, India

Antibacterial and antioxidant activities of methanol extract and fractions of Clerodendrum viscosum Vent. leaves

The present study was undertaken to evaluate antimicrobial and antioxidant activities of methanol extract as well as n-hexane, chloroform, ethyl acetate and aqueous fractions of Clerodendrum viscosum Vent. leaves. The antimicrobial activity, total phenolics and flavonoids were determined by agar-well diffusion, Folin–Ciocalteu and aluminum chloride colorimetric methods, respectively The antioxidant potential was evaluated by using various in vitro methods such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), nitric oxide and hydrogen peroxide radical scavenging assays. The maximum inhibition zones for S. aureus, B. subtilis, E. coli, K. pneumoniae and P. aeruginosa were 27, 25, 28, 23 and 22 mm for ethyl acetate fraction, respectively, whereas n-hexane fraction of methanol extract showed the weakest antimicrobial activity with inhibitory effect against B. subtilis and K. pneumoniae. The total phenolic content (TPC) in ethyl acetate fraction (87.28 mg GAE/g) was highest, followed by methanol extract (76.75 mg GAE/g), aqueous fraction (61.36 mg GAE/g) and chloroform fraction (28.46 mg GAE/g), respectively. The total flavonoid content (TFC) was in order of ethyl acetate fraction (77.48 mg quercetin equivalent/ g) > methanol extract (64.56 mg quercetin equivalent/ g) > aqueous fraction (49.66 mg quercetin equivalent/g) > chloroform fraction (32.85 mg quercetin equivalent/ g). The ethyl acetate fraction of methanol extract of C. viscosum leaves showed moderate free radical scavenging activity as evidenced by IC50 values in DPPH (64.51μg/mL), ABTS (28.32μg/mL), nitric oxide (80.02μg/mL) and H2O2 (77.45μg/mL) scavenging assays. The results of present study showed that C. viscosum leaves possess high phenolic, flavonoid contents, potential antimicrobial and antioxidant activity and could be a potential source of natural antioxidant that could have great importance as therapeutic agents in preventing or slowing the progress of ageing and age associated oxidative stress related degenerative diseases