Hormones andCuscuta development: interaction of cytokinin and indole-3-acetic acid in the growth and curvature of subapical stem segments

Cuscuta stem (vines) exhibits two modes of growth—longitudinal elongation forming free-hanging vines, or coiling growth to twine around the host. The elongation zone of free-hanging vine extended up to 160 mm from the stem apex and in vivo growth rate (during 8 h of growth) was maximal in the 20-to-40-mm region. While gibberellic acid (GA₃) or fusicoccin (FC) could maintain (GA₃) or enhance (FC) the growth rate of apical (10 or 25 mm) segments, indole-3-acetic acid (IAA) (10 M) induced growth only in subapical (5–160 mm) segments. In vitro growth rate induced by IAA (10 M) was similar to the in vivo growth rate up to 40 mm. Thereafter, up to 100 mm, IAA induced growth rate exceeded in vivo growth. p ]Subapical segments (13 mm) from 5- to 40-mm regions responded to a cytokinin (BA, Z, or iP) or to low IAA (0.1 M) with curved growth, whereas the segments grew straight in the presence of high IAA (10 M). Curvature (measured as the angle subtended at the center of the circle of which the segment formed an arc) induced by BA and low (0.1 M) IAA was greater than either added separately. Besides, segments induced to curve in BA + low-IAA solution could be made to straighten out by transferring to a solution containing high IAA (10 M) with or without BA. Thus in vivo patterns of straight and coiling growth could be mimicked reversibly in vitro by adjusting the relative concentrations of cytokinin and auxin; low auxin and cytokinin induced coiling growth, whereas high auxin and cytokinin induced straight growth. p ]Beyond 40 mm, BA had no growth-promoting or curvative-inducing effect.Cuscuta vine segments thus showed sequential sensitivity to applied hormones, the apical region (0–25 mm) to GA₃, the subapical (5–40 mm) region to BA and IAA and the region beyond (40–160 mm) to IAA alone.     

A low-cost,high-throughput microfluidic nano-culture platform for functional metagenomics

Functional metagenomics is an attractive culture-independent approach for functional screening of diverse microbiomes to identify known and novel genes. Since functional screening can involve sifting through tens of thousands of metagenomic library clones, an easy high-throughput screening approach is desirable. Here, we demonstrate a proof-of-concept application of a low-cost, high-throughput droplet based microfluidic assay to the selection of antibiotic resistance genes from a soil metagenomic library. Metagenomic library members encapsulated in nanoliter volume water-in-oil droplets were printed on glass slides robotically, and cell growth in individual drops in the presence of ampicillin was imaged and quantified to identify ampicillin-resistant clones. From the hits, true positives were confirmed by sequencing and functional validation. The ease of liquid handling, ease of set-up, low cost, and robust workflow makes the droplet-based nano-culture platform a promising candidate for screening and selection assays for functional metagenomic libraries.     

De novo design and Rosetta-based assessment of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike receptor binding domain (RBD)

The continued emergence of new SARS-CoV-2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high-affinity Ab variable regions (Fv) through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn-2.0. Subsequently, we carried out computational affinity maturation of the designed variable regions through amino acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9-mer library of “human Abs” based on a human string content score. We generated 106 different antibody designs and reported in detail on the top five that trade-off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn-2.0 using a Rosetta-based approach. We used Rosetta SnugDock for local docking of the designs to evaluate their potential to bind the spike RBD and performed “forward folding” with DeepAb to assess their potential to fold into the designed structures. Ultimately, our results identified one designed Ab variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of Abs that can quickly be adapted to target spike epitopes of emerging SARS-CoV-2 variants or other antigenic targets.     

Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein

Staphylococcus aureus pathogenesis depends on a specialized protein secretion system (ESX-1) that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB, small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary-structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacterium tuberculosis, suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and/or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins.     

Mechanics of a mosquito bite with applications to microneedle design

The mechanics of a fascicle insertion into the skin by a mosquito of the type aedes aegypti has been studied experimentally using high-speed video (HSV) imaging, and analytically using a mathematical model. The fascicle is a polymeric microneedle composed of a ductile material, chitin. It has been proposed that the mosquito applies a non-conservative follower force component in addition to the Euler compressive load in order to prevent buckling and penetrate the skin. In addition, the protective sheath surrounding the fascicle (labium) provides lateral support during insertion. The mechanics model presented approximates the fascicle as a slender column supported on an elastic foundation (labium) subjected to non-conservative (Beck) and conservative Euler loads simultaneously at the end. Results show that the lateral support of the fascicle provided by the labium is essential for successful penetration by increasing the critical buckling load by a factor of 5. The non-conservative follower force application increases the buckling load by an additional 20% and may or may not be necessary for successful penetration. Experimental results showing the importance of the labium have been cited to validate the model predictions, in addition to the video observations presented in this work. This understanding may be useful in designing painless needle insertion systems as opposed to miniaturized hypodermic needles.     

Computational modeling of morphogenesis regulated by mechanical feedback

Mechanical forces cause changes in form during embryogenesis and likely play a role in regulating these changes. This paper explores the idea that changes in homeostatic tissue stress (target stress), possibly modulated by genes, drive some morphogenetic processes. Computational models are presented to illustrate how regional variations in target stress can cause a range of complex behaviors involving the bending of epithelia. These models include growth and cytoskeletal contraction regulated by stress-based mechanical feedback. All simulations were carried out using the commercial finite element code ABAQUS, with growth and contraction included by modifying the zero-stress state in the material constitutive relations. Results presented for bending of bilayered beams and invagination of cylindrical and spherical shells provide insight into some of the mechanical aspects that must be considered in studying morphogenetic mechanisms.     

Water deficit induced oxidative damage in tea (Camellia sinensis) plants

When the tea (Camellia sinensis) leaf water potential was -1.1 MPa (Moderate water deficit), there was 58% inhibition of photosynthesis accompanied by increased zeaxanthin, malondialdehyde, oxidized proteins and superoxide dismutase activity. When the leaf water potential was -2MPa (severe water deficit), there was nearly complete inhibition of photosynthesis apart from a decrease in chlorophylls, beta-carotene, neoxanthin and lutein. Water deficit at this level caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde and oxidized proteins. It is inferred that, in tea plants, the increase in catalytic Fe and the decrease in antioxidant protection may be involved in the oxidative damage caused by severe water deficit, but not necessarily in the incipient stress induced by moderate water deficit.     

Commissioning and Acceptance Testing of the existing linear accelerator upgraded to volumetric modulated arc therapy

Aim

The RapidArc commissioning and Acceptance Testing program will test and ensure accuracy in DMLC position, precise dose-rate control during gantry rotation and accurate control of gantry speed.

Background

Recently, we have upgraded our linear accelerator capable of performing IMRT which was functional from 2007 with image guided RapidArc facility. The installation of VMAT in the existing linear accelerator is a tedious process which requires many quality assurance procedures before the proper commissioning of the facility and these procedures are discussed in this study.

Materials and methods

Output of the machine at different dose rates was measured to verify its consistency at different dose rates. Monitor and chamber linearity at different dose rates were checked. DMLC QA comprising of MLC transmission factor measurement and dosimetric leaf gap measurements were performed using 0.13 cm³ and 0.65 cm³ Farmer type ionization chamber, dose 1 dosimeter, and IAEA 30 cm × 30 cm × 30 cm water phantom. Picket fence test, garden fence test, tests to check leaf positioning accuracy due to carriage movement, calibration of the leaves, leaf speed stability effects due to the acceleration and deceleration of leaves, accuracy and calibration of leaves in producing complex fields, effects of interleaf friction, etc. were verified using EDR2 therapy films, Vidar scanner, Omnipro accept software, amorphous silicon based electronic portal imaging device and EPIQA software.^1–8

Results

All the DMLC related quality assurance tests were performed and evaluated by film dosimetry, portal dosimetry and EPIQA.⁷

Conclusion

Results confirmed that the linear accelerator is capable of performing accurate VMAT.     

Insights from an awareness survey on Oral Submucous Fibrosis (OSMF)

Oral Submucous Fibrosis (OSMF) is characterized by Juxta-epithelial inflammation with oedema, fibroblasts, neutrophil and eosinophil inflammatory infiltrate. Therefore, it is of interest to gain insights from an awareness survey on ORAL SUBMUCOUS FIBROSIS (OSMF). The sample size was 100 patients who had participated in this survey in Tamilnadu, India where 93% of the subjects used tobacco products. 81% of the patients were not aware of the malignancy caused by OSMF. A significant number of males use more tobacco than females (p value = 0.006 (<0.05) in the sample dataset. Thus, there is a need to create awareness on OSMF among the general population to combat the associated diseases.     

Apoptolysis: a less understood concept in the pathogenesis of Pemphigus Vulgaris

Pemphigus Vulgaris (PV) is a severe autoimmune disease characterized by supra-basal blisters in the skin and mucous membranes of a wide range of mammals, including humans. It not only affects the skin but also has severe oral manifestations. It has been stated that auto-antibodies are produced, for unknown reasons, which are directed against desmogleins present on the epithelium and thus leads to acantholysis and intraepithelial blistering. But the exact mechanism is still not completely understood. Here we would like to shed light on a new pathologic mechanism i.e., apoptolysis, which emphasizes that apoptotic enzymes contribute to acantholysis development both in terms of molecular events and chronologic sequence. A possible role of apoptolysis has been discussed in purview of PV.