Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex^?. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.     

SARS-CoV-2 variants: A double-edged sword?

Since the worldwide emergence of the COVID-19 outbreak, there have been international concerns about the possible viral evolution into variants with underlying mutations that may contribute to their increased transmissibility, disease severity, risk of death, and their potential escape from the immune response or may even lead to its extinction. Rigorous surveillance has revealed the variants harboring mutations in the spike protein, the main target of neutralizing antibodies generated through vaccination or herd immunity. In this review, we have highlighted major SARS-CoV-2 variants as well as other local strains along with their specific mutations, suspected changes in their characteristics, and their impact on the current pandemic and vaccine efficacy. We have also emphasized the need to develop widely protective interventions to curb further transmission of variants.     

Structure of Polymorphic Stomata in Canella winterena (L.) Geartn. (Canellaceae)

The presence of 10 types of normal stomata along with 4 subtypes, one intermediate type between stephanocytic and hemiparacytic and some abnormal stomatal structures both in the vegetative and floral organs of Canella winterena (L.) Geartn. (Canellaceae) is reported here. The epidermal cells are polygonal, isodiametric in surface view with straight anticlinal walls. The stomata are amphibrachyparacytic, anomocytic, anisocytic, brachyparacytic, brachyparatetracytic, cyclocytic, hemiparacytic, laterocytic, holoparacytic, stephanocytic, one intermediate type between stephanocytic and hemiparacytic, etc. Abnormalities like contiguous stomata, different types of twin stomata and cytoplasmic bridge between adjacent stomata, stomata with single guard cell, single guard cell with pore juxtaposed with normal stoma are also found. The presence of giant stomata is a significant finding. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of aging on male reproduction in zebrafish (Danio rerio)

The study was designed to test the hypothesis that male aging is associated with a change in reproductive function in the zebrafish. Young (290 ± 37 d) and older (911 ± 48 d) males were combined with females (604 ± 24 d) to test the effect of male age on the number and fertility of eggs laid by their mates. 48% of breeding trials with young males and 25% of the trails with older males resulted in egg deposition. Although young males were associated with significantly more successful breeding attempts than older males, number of eggs laid per clutch, number and percent of fertilized eggs and the number and percent living embryos were not statistically different between young and older males. These data suggest that male aging is associated with altered reproductive behavior and/or female response but not in sperm quality per se. Consistent with this interpretation were the findings that percent motility and sperm motility characteristics did not differ between sperm from young and older males as assessed by computer-assisted sperm analysis. However, older males contained higher quantities of extractable sperm than did young males, perhaps associated with fewer successful breeding attempts. Age-related effects on male reproductive in the zebrafish may therefore be a consequence of behavioral or morphological features that play a role in female mate choice and/ or male sexual response.     

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.     

Interaction of recombinant CanPIs with Helicoverpa armigera gut proteases reveals their processing patterns,stability and efficiency

Six diverse representative Capsicum annuum (common name: hot pepper; Solanaceae) protease inhibitor genes, viz CanPI‐5, ‐7, ‐13, ‐15, ‐19, and 22 comprising 1–4 inhibitory repeat domains (IRDs), were cloned and expressed in Pichia pastoris. The recombinant proteins were evaluated for their interactions with bovine trypsin, chymotrypsin, and Helicoverpa armigera gut proteases (HGP) using electrophoretic (native and denaturing) and mass spectrometric (MALDI‐TOF‐MS in combination with intensity fading assays) techniques. These techniques allow qualitative and semiquantitative analysis of multiple and processed IRDs of purified recombinant Capsicum annuum proteinase inhibitor (rCanPI) proteins. rCanPIs showed over 90% trypsin inhibition, varying chymotrypsin inhibition depending on the number of respective IRDs and over 60% inhibition of total HGP. rCanPI‐15 that has only one IRD showed exceptionally low inhibition of these proteases. Interaction studies of rCanPIs with proteases using intensity fading‐MALDI‐TOF‐MS revealed gradual processing of multi‐IRD rCanPIs into single IRD forms by the action of HGP at the linker region, unlike their interactions with trypsin and chymotrypsin. Intensity fading‐MALDI‐TOF‐MS assay showed that CanPI‐13 and ‐15, possessing single IRD and expressed predominantly in stem tissue are degraded by HGP; indicating their function other than defense. In vitro and in vivo studies on rCanPI‐5 and ‐7 showed maximum inhibition of HGP isoforms and their processed IRDs were also found to be stable in the presence of HGP. Even single amino acid variations in IRDs were found to change the HGP specificity like in the case of HGP‐8 inhibited only by IRD‐12. The presence of active PI in insect gut might be responsible for changed HGP profile. rCanPI‐5 and ‐7 enhanced HGP‐7, reduced HGP‐4, ‐5, ‐10 expression and new protease isoforms were induced. These results signify isoform complexity in plant PIs and insect proteases.     

Contextual correlates of child malnutrition in rural Maharashtra

This paper examines the role of observed contextual factors like topography, development and literacy on severe malnutrition among social groups in rural Maharashtra based on the Reproductive and Child Health District Level Household Survey (RCH-DLHS) Round II (2002-04) data. Multilevel modelling techniques were applied in order to examine the district-wise variations in severe malnutrition associated with the characteristics of the places (contextual effects), as the relationships with the type of people (compositional effects) have already been well established. The results show that developmental aspects such as road connectivity, community literacy, toilet facilities and household standard of living contribute positively to the status of severe malnutrition. Also, the scheduled tribe, aboriginal underprivileged group are more at risk of severe malnourishment due to a lack of proper development, poor awareness about maintaining and enhancing the nutritional value of food and lack of hygiene and sanitation as compared with the scheduled castes, another aboriginal group.     

Characterization of two midgut proteinases of Helicoverpa armigera and their interaction with proteinase inhibitors

Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants.     

Subunits of the translocon interact with components of the oligosaccharyl transferase complex

Following initiation of translocation across the membrane of the endoplasmic reticulum via the translocon, polypeptide chains are N-glycosylated by the oligosaccharyl transferase (OT) enzyme complex. Translocation and N-glycosylation are concurrent events and would be expected to require juxtaposition of the translocon and the OT complex. To determine whether any of the subunits of the OT complex and translocon mediate interactions between the two complexes, we initiated a systematic study in budding yeast using the split-ubiquitin approach. Interestingly, the OT subunit Stt3p was found to interact only with Sec61p, whereas another OT subunit, Ost4p, was found to interact with all three components of the translocon, Sec61p, Sbh1p, and Sss1p. The OT subunit Wbp1p was found to interact very strongly with Sec61p and Sbh1p and weakly with Sss1p. Other OT subunits, Ost1p, Ost2p, and Swp1p were found to interact with Sec61p and either Sbh1p or Sss1p. Ost3p exhibited a weak interaction with Sec61p and Sbh1p, whereas Ost5p and Ost6p interacted very weakly with Sec61p and failed to interact with Sbh1p or Sss1p. We were able to confirm these split-ubiquitin findings by a chemical cross-linking technique. Based on our findings using these two techniques, we conclude that the association of these two complexes is stabilized via multiple protein-protein contacts. Based on extrapolation of the structural parameters of the crystal structure of the prokaryotic Sec complex to the eukaryotic complex, we propose a working model to understand the organization of the translocon-OT supercomplex.     

Disruption of methylarginine metabolism impairs vascular homeostasis

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.