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Ethnopharmacological relevance For millennia, Artemisia annua L. was used in Southeast Asia to treat “fever”. This medicinal plant is effective against multiple pathogens and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria caused by Plasmodium parasites. Aim of the study The SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants. Materials and methods Using Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua L. extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1). Results In addition to being effective against the original wild type (WT) WA1, A. annua cultivars A3, BUR, MED, and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3 to 8.4 μM and 1.4–25.0 μM, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0 to 67.7 μg DW and 59.5–160.6 μg DW, respectively. Cell toxicity was insignificant at a leaf dry weight of ≤50 μg in the extract of any cultivar. Conclusions Results suggest that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines., Graphical abstract Image 1

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2., Graphical abstract, Banach et al. report a SARS-CoV-2 neutralizing antibody along with genetic, structural, and functional features of public antibody responses targeting SARS-CoV-2. These data reveal how structural interactions with the SARS-CoV-2 receptor-binding domain correlate with viral neutralization and demonstrate the importance of native antibody heavy:light pairings in convergent antibody responses.

Ethnopharmacological relevance Artemisia annua L. has been used for millennia in Southeast Asia to treat “fever”. Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. Aim of the Study SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. Materials and Methods Using Vero E6 and Calu-3 cells, we measured anti viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated. Results Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 μM, 0.01-0.14 μg, and 23.4-57.4 μg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet the hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 μM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 μM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. Conclusions A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections., Graphical abstract Image 1

Adequate health care facilities are not available for people of low resource settings in India. It is even worse when it comes to the monitoring of the health of pregnant women in villages. In this paper we discuss about the integrated healthcare system which has both vital parameter monitoring and ultrasound scanning, particularly for pregnant women in rural areas. We present in depth about the significance of vital parameter measurements in pregnant women and the system we designed for this purpose and the experimental setup. The measured parameters are stored in SD card by the healthcare workers who carry the portable system to villages. This SD card is taken to the nearest primary health care center for diagnosis of any pre-natal disorders by medical experts. The same is reported back to the pregnant women in case of any treatment is required. The measured parameter values are also provided in this paper. Such a system would help in global effort for reduction in child and mother mortality rates in low resource settings, identification of prenatal disorders and treatment at right time.

Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins.

Picture compression could be a wide dynamic investigation space. In the present time, a few redesigns are made in compression systems and more advancements are imperative since the excitement for limit and transmission of visual data is perseveringly on an augmentation. This paper analyses compression utilizing visual redundancy and examines a couple of customary schemes used in picture compression, couple of present day compression frameworks towards visual quality, execution, method predictability and intricacy as opposed to pixel-wise dependability. The paper demonstrates the correlation of transforms: Discrete Cosine Transform, Haar Wavelet Transform and Karhunen-Loeve Transform and picture compression schemes: JPEG and BPG for execution in an exceedingly still-compression framework and highlight the upsides of those transforms and schemes in today's ways. The paper analyses DCT, KLT and HWT in the light of PSNR, compression extent and time intricacy and shows that DCT is the perfect among the considered transforms. The paper likewise exhibits that BPG is that the best scheme strengthened by its compression quality.

Autonomic dysfunction is a serious complication of diabetes and can lead to cardiovascular abnormalities and premature death. It was recently proposed that autonomic dysfunction is triggered by oxidation-mediated inactivation of neuronal nicotinic acetylcholine receptors (nAChRs), impairing synaptic transmission in sympathetic ganglia and resulting in autonomic failure. We investigated whether the receptor for advanced glycation end products (RAGE) and its role in the generation of reactive oxygen species (ROS) could be contributing to the events that initiate sympathetic malfunction under high glucose conditions. Using biochemical, live imaging and electrophysiological tools we demonstrated that exposure of sympathetic neurons to high glucose increases RAGE expression and oxidative markers, and that incubation with RAGE ligands (e.g. AGEs, S100 and HMGB1) mimics both ROS elevation and nAChR inactivation. In contrast, co-treatment with either antioxidants or an anti-RAGE IgG prevented the inactivation of nAChRs. Lastly, a role for RAGE in this context was corroborated by the lack of sensitivity of sympathetic neurons from RAGE knock-out mice to high glucose. These data define a pivotal role for RAGE in initiating the events associated with exposure of sympathetic neurons to high glucose, and strongly support RAGE signaling as a potential therapeutic target in the autonomic complications associated with diabetes.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation.

Angiogenesis is a critical player in many pathologic processes, especially in neoplasms. Unregulated angiogenesis is thought to be indispensable in tumor growth and metastasis. Angiogenesis is the growth of new blood vessels from preexisting vasculature. The growing tumor mass requires an adequate amount of oxygen and nutrients which are supplied by complex network of tumor blood microvessels. The identification of molecules involved in the regulation of angiogenesis may provide new possibilities for the development of drugs suitable for inhibition of angiogenesis.

A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only α-helix 5 of domain I along with adjacent helices α-4 or α-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of α-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.

Capillary Haemangioma is a benign vascular tumour characterized by proliferation of blood vessels that are primarily reported to be a developmental hamartomatous lesion of infancy and childhood. Pyogenic granuloma is a non-neoplastic benign lesion found in the oral cavity having a striking predilection for occurrence in the gingiva. The present case report is an atypical presentation of capillary haemangioma on gingiva which is considered to be extremely rare. The lesion in this case was clinically diagnosed as pyogenic granuloma but histopathologically as capillary haemangioma. These lesions present as a diagnostic dilemma to the clinician and can lead to serious complications if not carefully managed.

The effect of fluoride treatment on the expression of a panel of osteogenic and stress markers in Stage 55 premetamorphic Xenopus larvae was examined at the precise onset of replacement of the larval cartilaginous skeleton with bone. A dosing regimen of 10 mmol/L sodium fluoride over 8 days was followed, during which time larvae developed to Stage 58, when the process of progressive ossification takes place in the vertebral column and membranous bones of the skull, pelvic, and pectoral girdles and portions of the appendicular skeleton. Markers of bone formation, including COL1A1, the transcription factors Osterix, RUNX2-II, and matrix metalloproteinases MMP1 and MMP13, decreased relative to age-matched controls, though the osteoblast marker BGLAP was not significantly altered. Expression of the pro-osteoclastogenic factor RANKL decreased, whereas expression of the anti-osteoclastogenic factor osteoprotegerin increased. Altered expression of oxidative stress markers, with the exception of superoxide dismutase, was generally not observed. These data demonstrate the potent effects of fluoride on the expression of factors required for osteoblast and osteoclast differentiation, as well as on the expression of osteoblast products, including MMP1 and collagen. Importantly, these effects were observed in the absence of significant changes in the expression of oxidative stress markers. The results provide the first molecular insights into the mechanisms underlying skeletal fluorosis in a whole organism developmental model.

Immobilized metal affinity chromatography (IMAC) is widely used for the production of recombinant proteins for a variety of applications; however, a number of challenges are typically encountered by researchers depending on the properties of the specific proteins in question. Here, we describe technical issues we have encountered in production of recombinant zinc finger nucleic acid-binding proteins by IMAC intended for detailed and accurate in vitro analysis. The process encountered leading to a modified IMAC protocol for effective production of high-purity, native zinc finger nucleic acid-binding proteins is described in detail. The parameters with respect to solubility, lysis and redox conditions, removal of residual metal ions with chelating agents, and renaturation in the presence of divalent metal cations are described. These procedures have been extended to production of a wide array of RNA-binding proteins in our laboratory and would be relevant to a number of protein purification applications.

The mosquitocidal crystal protein, Cry19Aa, from Bacillus thuringiensis ssp. jegathesan, has high toxicity to Anopheles stephensi and Culex pipiens but is less toxic to Aedes aegypti. To study the functional role of putative domain II and surface residues in mosquito toxicity, 16 alanine substitution mutations were introduced into Cry19Aa. All mutant constructs were expressed as 65-kDa protoxins and subsequently digested by trypsin to produce further fragmented polypeptides of 40 and 25 kDa. With chymotrypsin, however, most protoxins were digested to 60 kDa and minor bands. The circular dichroism spectra of the chymotrypsin-activated toxins of Cry19Aa and muteins, Y324A, W357A, Y412A, Y414A, W416A, D418A and F485A indicated that there was no significant variation in their structure. In mosquito bioassays, Y324A, W357A, Y410A, W416A, D418A and F485A muteins showed substantial reductions in mosquitocidal activity toward A. aegypti and C. pipiens. These muteins also showed reduced competition with wild-type fluorescein 5-isothiocyanate-labeled Cry19Aa for binding to C. pipiens brush border membrane vesicles. These data suggest that the reduction of toxicity was a result of the reduced binding affinity. From these studies we have identified loop residues of domain II that are important in toxicity and receptor binding to Culex larval midgut.

The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.