Electronic field effect detection of SARS-CoV-2 N-protein before the onset of symptoms.

As part of the efforts to contain the pandemic, researchers around the world have raced to develop testing platforms to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the Coronavirus disease 2019 (COVID-19). Within the different detection platforms studied, the field effect transistor (FET) is a promising device due to its high sensitivity and fast detection capabilities. In this work, a graphene-based FET which uses a boron and nitrogen co-doped graphene oxide gel (BN-GO gel) transducer functionalized with nucleoprotein antibodies, has been investigated for the detection of SARS-CoV-2 nucleocapsid (N)-protein in buffer. This biosensor was able to detect the viral protein in less than 4 min, with a limit of detection (LOD) as low as 10 ag/mL and a wide linear detection range stretching over 11 orders of magnitude from 10 ag/mL-1 µg/mL. This represents the lowest LOD and widest detection range of any COVID-19 sensor and thus can potentially enable the detection of infected individuals before they become contagious. In addition to its potential use in the COVID-19 pandemic, our device serves as a proof-of-concept of the ability of functionalized BN-GO gel FETs to be used for ultrasensitive yet robust biosensors.

Cerebral Venous Thrombosis in COVID-19: A New York Metropolitan Cohort Study.

BACKGROUND AND PURPOSE: Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infection is associated with hypercoagulability. We sought to evaluate the demographic and clinical characteristics of cerebral venous thrombosis among patients hospitalized for coronavirus disease 2019 (COVID-19) at 6 tertiary care centers in the New York City metropolitan area. MATERIALS AND METHODS: We conducted a retrospective multicenter cohort study of 13,500 consecutive patients with COVID-19 who were hospitalized between March 1 and May 30, 2020. RESULTS: Of 13,500 patients with COVID-19, twelve had imaging-proved cerebral venous thrombosis with an incidence of 8.8 per 10,000 during 3 months, which is considerably higher than the reported incidence of cerebral venous thrombosis in the general population of 5 per million annually. There was a male preponderance (8 men, 4 women) and an average age of 49 years (95% CI, 36-62 years; range, 17-95 years). Only 1 patient (8%) had a history of thromboembolic disease. Neurologic symptoms secondary to cerebral venous thrombosis occurred within 24 hours of the onset of the respiratory and constitutional symptoms in 58% of cases, and 75% had venous infarction, hemorrhage, or both on brain imaging. Management consisted of anticoagulation, endovascular thrombectomy, and surgical hematoma evacuation. The mortality rate was 25%. CONCLUSIONS: Early evidence suggests a higher-than-expected frequency of cerebral venous thrombosis among patients hospitalized for COVID-19. Cerebral venous thrombosis should be included in the differential diagnosis of neurologic syndromes associated with SARS-CoV-2 infection.

An ultrasensitive heart-failure BNP biosensor using B/N co-doped graphene oxide gel FET.

Heart failure (HF) is the number one cause of death in the world. B-type natriuretic peptide (BNP) is a recognized biomarker for HF and can be used for early detection. Field effect transistor (FET) biosensors have the ability to sense BNP in much shorter times than conventional clinical studies. The lowest limit of detection (LOD) of state-of-the-art HF FET biosensors is 100 fM and detection ranges are short, being less than 4 orders of magnitude. In this work, a B/N co-doped graphene oxide (GO) gel (BN-GO) was used as the channel material in an FET biosensor targeting BNP. The sensor was able to sense BNP in as little as 2 min, with an LOD as low as 10 aM and a wide linear detection range of 10 aM-1 µM, stretching over 11 orders of magnitude. The biosensor showed great selectivity and minimal response towards K+ and OH- ions and the human epidermal growth factor receptor (HER2) protein. This biosensor serves as a proof-of-concept of the ability of BN-GO gel FETs to be used for ultrasensitive biosensors.

Synergistic performance of triggered drug release and photothermal therapy of MCF7 cells based on laser activated PEGylated GO + DOX.

Graphene oxide is used as a singular 2D nano-carrier in cancer therapy. Here, graphene oxide is used as a hybrid chemo-drug graphene oxide (GO) + doxorubicin (DOX), mainly due to its unique chemical and optical properties. The laser triggers GO + DOX for selective drug delivery to optimize the drug release. The characterization of GO is investigated in terms of laser properties at 808 nm. Furthermore, the laser activates GO + DOX compounds to treat MCF7 cancerous cells. The drug release strongly depends on the temperature rise that mainly effects on the viability of the cancerous cells of interest. DOX simultaneously acts as a chemo-drug and as an optical fluorescent agent, whereas GO performs as an efficient photothermal nano-carrier. In fact, the GO-DOX hybrid drug demonstrates multifunctional during malignant cell treatment. We have shown that the laser heating of GO enhances the release percentage up to a treatment yield of 90%. This arises from the synergistic nature of DOX and GO compounds in simultaneous chemo/photo thermal therapy. Furthermore, the fluorescence property of DOX is used to assess the GO uptake using confocal microscope imaging.

Effects of supplemental conjugated linoleic acids (CLA) on fresh and post-thaw sperm quality of Holstein bulls.

This study was designed to investigate the effects of feeding-protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top-dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post-thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer-assisted), viability (Eosin-Nigrosin), membrane integrity (hypo-osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA-fed group compared to control group. Also, in CLA-fed group, the proportion of post-thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA-fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen-thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post-thaw sperm quality of Holstein bulls.

Health effects of probiotics on the skin.

Skin is the largest organ of the body and is constantly exposed to physical, chemical, bacterial, and fungal challenges. It is well known that probiotics are helpful for specific disorders and different clinical studies have indicated that probiotics have special effects in cutaneous apparatus directly or indirectly that can be considerable from versatile aspects. Probiotic bacteriotherapy can have great potential in preventing and treating the skin diseases including eczema, atopic dermatitis, acne, and allergic inflammation or in skin hypersensitivity, UV-induced skin damage, wound protection, and as a cosmetic product. The current paper comprehensively reviews the different health effects of probiotics on the skin.

High-throughput search for new permanent magnet materials.

The currently highest-performance Fe-Nd-B magnets show limited cost-effectiveness and lifetime due to their rare-earth (RE) content. The demand for novel hard magnetic phases with more widely available RE metals, reduced RE content or, even better, completely free of RE metals is therefore tremendous. The chances are that such materials still exist given the large number of as yet unexplored alloy systems. To discover such phases, an elaborate concept is necessary which can restrict and prioritize the search field while making use of efficient synthesis and analysis methods. It is shown that an efficient synthesis of new phases using heterogeneous non-equilibrium diffusion couples and reaction sintering is possible. Quantitative microstructure analysis of the domain pattern of the hard magnetic phases can be used to estimate the intrinsic magnetic parameters (saturation polarization from the domain contrast, anisotropy constant from the domain width, Curie temperature from the temperature dependence of the domain contrast). The probability of detecting TM-rich phases for a given system is high, therefore the approach enables one to scan through even higher component systems with one single sample. The visualization of newly occurring hard magnetic phases via their typical domain structure and the correlation existing between domain structure and intrinsic magnetic properties allows an evaluation of the industrial relevance of these novel phases.

Incorporation of Lactobacillus casei in Iranian ultrafiltered Feta cheese made by partial replacement of NaCl with KCl.

Probiotic Iranian ultrafiltered Feta cheese was produced from ultrafiltration of milk with a volumetric concentration factor of 4.5:1. The heat-treated retentates were inoculated with 10(7) cfu of Lactobacillus casei LAFTI L26/mL. A mesophilic-thermophilic mixed culture of Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Streptococcus thermophilus was also used. Three percent (wt/wt) salt with different ratios of NaCl:KCl (100% NaCl, 50% NaCl:50% KCl, 75% NaCl:25% KCl, and 25% NaCl:75% KCl) were used in cheese formulation. The viability of L. casei was determined in treatments during the ripening period (90d at 5°C) within 15-d intervals. The pH, titratable acidity, and redox potential changes were monitored throughout the mentioned period. The mean pH drop rate, mean acidity increase rate, and mean redox potential increase rate were calculated at the end of the storage period. Also, total nitrogen, water-soluble nitrogen, lactic acid, and acetic acid concentrations, and syneresis and sensory characteristics of the product were measured during the mentioned period every 30d. The maximum viability of L. casei was observed within d 15 to 30 of the ripening period in the treatment containing the lowest amount of sodium. Addition of KCl enhanced syneresis. Cheeses with NaCl alone and with only 25% replacement by KCl have the highest sensory acceptability.

Biophysical coarse-grained modeling provides insights into transport through the nuclear pore complex.

The nuclear pore complex (NPC) is the gatekeeper of the nucleus, capable of actively discriminating between the active and inert cargo while accommodating a high rate of translocations. The biophysical mechanisms underlying transport, however, remain unclear due to the lack of information about biophysical factors playing role in transport. Based on published experimental data, we have established a coarse-grained model of an intact NPC structure to examine nucleocytoplasmic transport with refined spatial and temporal resolutions. Using our model, we estimate the transport time versus cargo sizes. Our findings suggest that the mean transport time of cargos smaller than 15 nm is independent of size, while beyond this size, there is a sharp increase in the mean transport time. The model confirms that kap-FG hydrophobicity is sufficient for active cargo transport. Moreover, our model predicts that during translocation, small and large cargo-complexes are hydrophobically attached to FG-repeat domains for 86 and 96% of their transport time, respectively. Inside the central channel FG-repeats form a thick layer on the wall leaving an open tube. The cargo-complex is almost always attached to this layer and diffuses back and forth, regardless of the cargo size. Finally, we propose a plausible model for transport in which the NPC can be viewed as a lubricated gate. This model incorporates basic assumptions underlying virtual-gate and reduction-of-dimensionality models with the addition of the FG-layer inside the central channel acting as a lubricant.

Distribution patterns of phosphorylated Thr 3 and Thr 32 of histone H3 in plant mitosis and meiosis.

Cell cycle dependent phosphorylation of conserved N-terminal tail residues of histone H3 has been described in both animal and plant cells. Through cytogenetic approaches using different plant species we show a detailed description of distribution patterns of phosphorylated histone H3 at either threonine 3 or threonine 32 in mitosis and meiosis. In meristematic cells of the large genome species Secale cereale, Vicia faba and Hordeum vulgare we have found that phosphorylation of both threonine residues begins in prophase, and dephosphorylation occurs in late anaphase. However, in the small genome species Arabidopsis thaliana dephosphorylation occurs at anaphase. In the first division of meiosis of species with large genomes phosphorylation of histone H3 at either threonine 3 or threonine 32 is seen first in diakinesis and extends to anaphase I, whereas in the second division these post-translational modifications are visible at metaphase II through anaphase II. While in A. thaliana dephosphorylation takes place at anaphase I and II. In all species analysed phosphorylated H3 at either threonine 3 or threonine 32 are distributed along the entire length of chromosomes during mitotic metaphase and metaphase I. In the second meiotic division threonine 3 phosphorylation is restricted to the pericentromeric domain, while phosphorylation of threonine 32 is widespread along chromosome arms of all species analysed.

Perforin lytic activity is controlled by calreticulin.

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.

Shutdown in protein synthesis due to the expression of mini-genes in bacteria.

Mutants of Escherichia coli partially deficient in peptidyl-tRNA hydrolase are killed by the expression of certain very short open reading frames (mini-genes), encoded by the wild-type bar regions of phage lambda. According to the current hypothesis, protein synthesis is shut off, and the host cells die, after essential tRNA species become sequestered due to abnormal translation termination (drop-off) of mini-gene-encoded peptides as peptidyl-tRNA. Here we study variants of bar mini-genes, both in vivo and in vitro, in order to identify the structural elements that influence this inhibition of protein synthesis. Three parameters were measured during the expression of these variants: the rates of normal translation termination, peptidyl-tRNA dissociation from the ribosome and hydrolysis of peptidyl-tRNA by peptidyl-tRNA hydrolase were measured. Previous observations that RRF, EF-G and RF3 stimulated drop-off were confirmed and extended; stimulation by these factors can reach 30-fold. Both factor-stimulated and spontaneous drop-off depended on the nature of the stop signal. The degree of inhibition of cell growth following induction of mini-gene expression could be accounted for in terms of a toxicity index comprising the three parameters above. Inhibition was greatly reduced in cells lacking RF3. Mini-genes with more efficient Shine/Dalgarno sequences killed cells even with normal peptidyl-tRNA hydrolase activity. It is proposed that the retranslation by ribosomes of mini-gene transcripts with efficient ribosome binding (Shine/Dalgarno) sequences strongly contributes to the inhibitory effects of mini-gene expression on protein synthesis.

Novel roles for classical factors at the interface between translation termination and initiation.

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.

Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes.

A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed.

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.

We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild-type ribosomes is much stronger than to their A-site. Addition of streptomycin dramatically reduces its affinity for the P-site. The S12 alternations make the P-site binding of peptidyl-tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild-type. We find that when binding of peptidyl-tRNA to the A-site is weak, then the affinity for the P-site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.

tRNA-ribosome interactions.

Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.

Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.

The binding stability of the aminoacyl-tRNA site (A-site), estimated from the dissociation rate constant kd, of AcPhe-Phe-tRNA(Phe) has been studied for wild-type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin-dependent (SmD) and streptomycin-pseudodependent (SmP) phenotypes], for error-prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error-inducing aminoglycosides streptomycin and neomycin. The AcPhe2-tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proof-reading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof-reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop-off of peptidyl-tRNAs from the A-site.