Epitope Mapping of Therapeutic Antibodies Targeting Human LAG3.

Lymphocyte activation gene 3 protein (LAG3; CD223) is an inhibitory receptor that is highly upregulated on exhausted T cells in tumors and chronic viral infection. Consequently, LAG3 is now a major immunotherapeutic target for the treatment of cancer, and many mAbs against human (h) LAG3 (hLAG3) have been generated to block its inhibitory activity. However, little or no information is available on the epitopes they recognize. We selected a panel of seven therapeutic mAbs from the patent literature for detailed characterization. These mAbs were expressed as Fab or single-chain variable fragments and shown to bind hLAG3 with nanomolar affinities, as measured by biolayer interferometry. Using competitive binding assays, we found that the seven mAbs recognize four distinct epitopes on hLAG3. To localize the epitopes, we carried out epitope mapping using chimeras between hLAG3 and mouse LAG3. All seven mAbs are directed against the first Ig-like domain (D1) of hLAG3, despite their different origins. Three mAbs almost exclusively target a unique 30-residue loop of D1 that forms at least part of the putative binding site for MHC class II, whereas four mainly recognize D1 determinants outside this loop. However, because all the mAbs block binding of hLAG3 to MHC class II, each of the epitopes they recognize must at least partially overlap the MHC class II binding site.

Progress in Ocular Drug Delivery: Challenges and Constraints.

The eye has several dynamic and static barriers in place to limit the entry of foreign substances including therapeutics. As such, efficient drug delivery, especially to posterior segment tissues, has been challenging. This chapter describes the anatomical and physiological challenges associated with ocular drug delivery before discussing constraints with regard to formulation parameters. Finally, it gives an overview of advanced drug delivery technologies with a specific focus on recently marketed and late-stage clinical trial products.

Cancer epithelia-derived mitochondrial DNA is a targetable initiator of a paracrine signaling loop that confers taxane resistance.

Stromal-epithelial interactions dictate cancer progression and therapeutic response. Prostate cancer (PCa) cells were identified to secrete greater concentration of mitochondrial DNA (mtDNA) compared to noncancer epithelia. Based on the recognized coevolution of cancer-associated fibroblasts (CAF) with tumor progression, we tested the role of cancer-derived mtDNA in a mechanism of paracrine signaling. We found that prostatic CAF expressed DEC205, which was not expressed by normal tissue-associated fibroblasts. DEC205 is a transmembrane protein that bound mtDNA and contributed to pattern recognition by Toll-like receptor 9 (TLR9). Complement C3 was the dominant gene targeted by TLR9-induced NF-κB signaling in CAF. The subsequent maturation complement C3 maturation to anaphylatoxin C3a was dependent on PCa epithelial inhibition of catalase in CAF. In a syngeneic tissue recombination model of PCa and associated fibroblast, the antagonism of the C3a receptor and the fibroblastic knockout of TLR9 similarly resulted in immune suppression with a significant reduction in tumor progression, compared to saline-treated tumors associated with wild-type prostatic fibroblasts. Interestingly, docetaxel, a common therapy for advanced PCa, further promoted mtDNA secretion in cultured epithelia, mice, and PCa patients. The antiapoptotic signaling downstream of anaphylatoxin C3a signaling in tumor cells contributed to docetaxel resistance. The inhibition of C3a receptor sensitized PCa epithelia to docetaxel in a synergistic manner. Tumor models of human PCa epithelia with CAF expanded similarly in mice in the presence or absence of docetaxel. The combination therapy of docetaxel and C3 receptor antagonist disrupted the mtDNA/C3a paracrine loop and restored docetaxel sensitivity.

Variety-specific transcript accumulation during reproductive stage in drought-stressed rice.

The divergence of natural stress tolerance mechanisms between species is an intriguing phenomenon. To study it in rice, a comparative transcriptome analysis was carried out in 'heading' stage tissue (flag leaf, panicles and roots) of Nagina 22 (N22; drought-tolerant) and IR64 (drought-sensitive) plants subjected to field drought. Interestingly, N22 showed almost double the number of differentially expressed genes (DEGs) than IR64. Many DEGs colocalized within drought-related QTLs responsible for grain yield and drought tolerance and also associated with drought tolerance and critical drought-related plant traits such as leaf rolling, trehalose content, sucrose and cellulose content. Besides, co-expression analysis of the DEGs revealed several 'hub' genes known to actively regulate drought stress response. Strikingly, 1366 DEGs, including 21 'hub' genes, showed a distinct opposite regulation in the two rice varieties under similar drought conditions. Annotation of these variety-specific DEGs (VS-DEGs) revealed that they are distributed in various biological pathways. Furthermore, 103 VS-DEGs were found to physically interact with over 1300 genes, including 32 that physically interact with other VS-DEGs as well. The promoter region of these genes has sequence variations among the two rice varieties, which might be in part responsible for their unique expression pattern.

Pyramiding of genes for grain protein content, grain quality, and rust resistance in eleven Indian bread wheat cultivars: a multi-institutional effort.

Improvement of grain protein content (GPC), loaf volume, and resistance to rusts was achieved in 11 Indian wheat cultivars that are widely grown in four different agro-climatic zones of India. This involved use of marker-assisted backcross breeding (MABB) for introgression and pyramiding of the following genes: (i) the high GPC gene Gpc-B1; (ii) HMW glutenin subunits 5 + 10 at Glu-D1 loci, and (iii) rust resistance genes, Yr36, Yr15, Lr24, and Sr24. GPC increased by 0.8 to 3.3%, although high GPC was generally associated with yield penalty. Further selection among high GPC lines allowed identification of progenies with higher GPC associated with improvement in 1000-grain weight and grain yield in the backgrounds of the following four cultivars: NI5439, UP2338, UP2382, and HUW468. The high GPC progenies (derived from NI5439) were also improved for grain quality using HMW glutenin subunits 5 + 10 at Glu-D1 loci. Similarly, progenies combining high GPC and rust resistance were obtained in the backgrounds of following five cultivars: Lok1, HD2967, PBW550, PBW621, and DBW1. The improved pre-bred lines developed following multi-institutional effort should prove a valuable source for the development of cultivars with improved nutritional quality and rust resistance in the ongoing wheat breeding programmes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01277-w.

Placental levels of polycyclic aromatic hydrocarbons (PAHs) and their association with birth weight of infants.

As an alarming group of pollutants, polycyclic aromatic hydrocarbons (PAHs) gather much public health concern not only because of their carcinogenic or co-carcinogenic risk but also by interfering with hormone systems or by causing oxidative damage, henceforth liable to toxic actions on reproduction. Accordingly, the present study was aimed to explore the association between in-utero exposure to PAHs by evaluating their placental levels and infant birth weight among 110 healthy and nonsmoking pregnant women. Placental tissue samples were collected instantly after delivery and were analyzed for the presence of sixteen Environmental Protection Agency (EPA) listed PAHs with the help of Gas chromatography equipped with flame ionization detector (GC-FID). Chrysene and benzo(k)fluoranthene were the predominant PAHs detected in tissue samples. To assess the source of origin of PAHs in placenta tissue samples, the ratio of low molecular weight PAHs to high molecular weight (∑LMW/∑HMW PAHs) was calculated, showing the predominance of pyrogenic sources of PAHs possibly responsible for the exposure of the studied population. Results of regression analysis demonstrated the inverse although not significant association of naphthalene (Nap), acenaphthylene (Acy), anthracene (Anth), pyrene (Pyr), benzo(b)fluoranthene (BbF), benzo(k)Fluoranthene (BkF), benzo(a)pyrene (BaP), indeno (123 cd pyrene (IcdP), dibenzo(ah)anthracene (DahA) and benzo(ghi)Perylene (BghiP) with birth weight of neonates. Additionally, the regression model lay light upon the significant association of fluoranthene (Fla) (coefficient= -1.41 gram, p < 0.05) to the depletion trend of birth weight after adjusting for potential covariates. These findings suggest the possible role of an environmental contaminants like PAHs on impairment of fetal growth.

Development of white-grained PHS-tolerant wheats with high grain protein and leaf rust resistance.

The present study involved incorporation of two major QTLs for pre-harvest sprouting tolerance (PHST) in an Indian wheat cultivar named Lok1, which happens to be PHS susceptible. For transfer of two QTLs, two independent programmes with two different donors (AUS1408, CN19055) were utilized. The recipient cv. Lok1 was crossed with each of the two donors, followed by a number of backcrosses. Each backcross progeny was subjected to foreground and background selections. KASP assay was also used for confirming the presence of PHST QTL. In one case, PHST QTL was later also pyramided with a gene for high grain protein content (Gpc-B1) and a gene for leaf rust resistance (Lr24). The MAS derived lines were screened for PHS using simulated rain chambers leading to selection of 10 PHST lines. Four of these advanced lines carried all the three QTL/genes and exhibited high level of PHST (PHS score 2-3) associated with significant improvement in GPC and resistance against leaf rust. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01234-z.

Ocular Distribution of Papaverine Using Non-aqueous Vehicles.

Papaverine, a poorly soluble opium alkaloid, has recently been shown to reduce retinal inflammation due to which it may have therapeutic application in the management of Leber's hereditary optic neuropathy. In this study, papaverine eyedrops based on medium chain triglycerides were prepared and the effect of diethyl glycol monoethyl ether (DGME) on their ocular distribution was evaluated using an ex vivo porcine eye model. The route of drug penetration was also studied by orienting the eye to expose either only the cornea or the sclera to the formulation. Furthermore, in vivo studies were performed to confirm ocular tolerability and evaluate ocular drug distribution. Our results showed increased papaverine concentrations in the cornea and sclera in the presence of DGME but with a slight reduction in the retina-choroid (RC) drug concentration when administered via the corneal route, suggesting that DGME enhances drug accumulation in the anterior ocular tissues but with little effect on posterior drug delivery. In vivo, the papaverine eyedrop with DGME showed good ocular tolerability with the highest drug concentration being observed in the cornea (1.53 ± 0.28 µg/g of tissue), followed by the conjunctiva (0.74 ± 0.18 µg/g) and sclera (0.25 ± 0.06 µg/g), respectively. However, no drug was detected in the RC, vitreous humor or plasma. Overall, this study highlighted that DGME influences ocular distribution and accumulation of papaverine. Moreover, results suggest that for hydrophobic drugs dissolved in hydrophobic non-aqueous vehicles, transcorneal penetration via the transuveal pathway may be the predominant route for drug penetration to posterior ocular tissues. Graphical abstract.

A novel leaf rust resistance gene introgressed from Aegilops markgrafii maps on chromosome arm 2AS of wheat.

KEY MESSAGE: A novel leaf rust resistance gene, LrM, introgressed from Aegilops markgrafii and mapped on chromosome 2AS using SSR- and SNP-based PCR markers will aid in broadening the genetic base of rust resistance in wheat. A new leaf rust resistance gene tentatively named LrM was introgressed from the diploid non-progenitor species Ae. markgrafii (2n = 2x = 14, genome CC) into common wheat using the nulli-5B mechanism. The introgression line ER9-700 showed a high degree of resistance against a wide spectrum of Puccinia triticina pathotypes. Genetic analysis was performed using the F1, F2, F2:3 and BC1F1 generations derived from the cross ER9-700/Agra Local. The results showed a single dominant gene for leaf rust resistance. The resistance gene LrM was mapped on chromosome arm 2AS using SSR- and SNP-based PCR markers. Preliminary mapping with SSR markers in the F2:3 population from the cross ER9-700/Agra Local identified two SSR markers flanking the LrM. SNPs were identified in the genomic region flanked by SSR markers, and SNP-based PCR markers were developed to construct the final map. Three SNP-based PCR markers co-segregated and mapped closest to the resistance gene at a distance of 2 cM. The gene LrM was distinguished from all the other genes designated and mapped on chromosome arm 2AS by molecular markers and rust reaction. All five markers used in the mapping amplified identical alleles in the donor Ae. markgrafii accession and introgression line ER9-700. The chromosomal location and rust reaction suggest that LrM is a novel leaf rust resistance gene that may be useful in broadening the genetic base of leaf rust resistance in wheat.

Unravelling the Role of Rhizospheric Plant-Microbe Synergy in Phytoremediation: A Genomic Perspective.

BACKGROUND: Accretion of organic and inorganic contaminants in soil interferes in the food chain, thereby posing a serious threat to the ecosystem and adversely affecting crop productivity and human life. Both endophytic and rhizospheric microbial communities are responsible for the biodegradation of toxic organic compounds and have the capability to enhance the uptake of heavy metals by plants via phytoremediation approaches. The diverse set of metabolic genes encoding for the production of biosurfactants and biofilms, specific enzymes for degrading plant polymers, modification of cell surface hydrophobicity and various detoxification pathways for the organic pollutants, plays a significant role in bacterial driven bioremediation. Various genetic engineering approaches have been demonstrated to modulate the activity of specific microbial species in order to enhance their detoxification potential. Certain rhizospheric bacterial communities are genetically modified to produce specific enzymes that play a role in degrading toxic pollutants. Few studies suggest that the overexpression of extracellular enzymes secreted by plant, fungi or rhizospheric microbes can improve the degradation of specific organic pollutants in the soil. Plants and microbes dwell synergistically, where microbes draw benefit by nutrient acquisition from root exudates whereas they assist in plant growth and survival by producing certain plant growth promoting metabolites, nitrogen fixation, phosphate solubilization, auxin production, siderophore production, and inhibition or suppression of plant pathogens. Thus, the plant-microbe interaction establishes the foundation of the soil nutrient cycle as well as decreases soil toxicity by the removal of harmful pollutants. CONCLUSION: The perspective of integrating genetic approach with bioremediation is crucial to evaluate connexions among microbial communities, plant communities and ecosystem processes with a focus on improving phytoremediation of contaminated sites.

Identification of modifiers of the plant height in wheat using an induced dwarf mutant controlled by RhtB4c allele.

In wheat, 25 Rht genes for dwarfness are known, which include both, GA-insensitive and GA-responsive genes. The GA-insensitive Rht genes have been widely used, although, their suitability under abiotic stress conditions has been questioned. This necessitated a search for alternative GA-responsive, spontaneous and induced dwarfing genes. We earlier reported an induced dwarf mutant ('dwarf mutant-3'; 44 cm), and the mutant allele was named Rht4c allele (2BL). This dwarf mutant was not suitable for cultivation due to its extra dwarf nature. Therefore, we searched for naturally occurring QTLs, which would modify the phenotype of 'dwarf-mutant-3' using 'mutant-assisted gene identification and characterization' (MAGIC) approach. For this purpose, the 'dwarf mutant-3' was crossed with a tall wheat cv. NP114 and homozygous mutant F2 plants (~ 25% of the progeny) were selected, which were phenotyped for plant height and genotyped using SSR markers. The data were utilized for QTL analysis and plant height. Six modifier QTLs were identified on chromosomes 2A, 2B and 4A. Two QTLs each on 2A and 2B were responsible for increase in plant height (described as 'enhancer modifiers'), whereas the remaining two QTLs located on 4A were responsible for reducing the plant height (described as 'suppressor modifiers'). It was hypothesized that the enhancer QTLs could be exploited for the development of semi-dwarf high yielding genotypes containing Rht4c allele. This is the first study of its kind in wheat demontsrating that the MAGIC approach could be used for identification of modifiers of the mutant phenotypes of other traits for wheat improvement.

Association Between Placental Polycyclic Aromatic Hydrocarbons (PAHS), Oxidative Stress, and Preterm Delivery: A Case-Control Study.

Polycyclic aromatic hydrocarbons are known to disturb the antioxidant defense system, which may indirectly contribute to induction of early pregnancy in women. Therefore, the present investigation was designed to offer preliminary information about exposure to PAHs by estimating their placental levels and its association with oxidative stress as well as with preterm birth. Placenta tissue samples were drawn after delivery from 84 healthy pregnant women, recruited at a local nursing home of Agra, India, and levels of PAHs were quantified by gas chromatograph equipped with flame ionization detector. To evaluate redox status biomarkers, malondialdehyde (MDA) and glutathione (GSH) were determined in placenta tissue. Significantly elevated levels of benzo(a)pyrene and MDA while decreasing trend of GSH was found in women with preterm delivery group (study) than women with a full-term delivery group (control). Results demonstrated higher, but statistically insignificant (p > 0.05), levels of naphthalene, anthracene, fluorene, pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, indeno[1,2,3-cd]pyrene, dibenzo(ah)anthracene, and benzo(ghi)perylene in the study group than the control group. However, higher and lower molecular weight PAHs showed significant correlation for the depletion trend of GSH sights upon an example of oxidative stress mechanism. Because of limited statistical power and absence of controlled confounders, this study does not provide an ample involvement of PAHs with preterm delivery but increased MDA and decreased GSH in cases than controls gives the possible contribution of PAHs to early delivery.

Fruit preferential activity of the tomato RIP1 gene promoter in transgenic tomato and Arabidopsis.

Isolation and functional characterization of tissue- and stage-specific gene promoters is beneficial for genetic improvement of economically important crops. Here, we have characterized a putative promoter of a ripening-induced gene RIP1 (Ripening induced protein 1) in tomato. Quantification of the transcript level of RIP1 showed that its expression is fruit preferential, with maximum accumulation in red ripe fruits. To test the promoter activity, we made a reporter construct by cloning 1450 bp putative RIP1 promoter driving the GUS (ß-glucuronidase) gene expression and generated stable transgenic lines in tomato and Arabidopsis. Histochemical and fluorometric assays validated the fruit-specific expression of RIP1 as the highest GUS activity was found in red ripe tomatoes. Similarly, we detected high levels of GUS activity in the siliques of Arabidopsis. On the contrary, weak GUS activity was found in the flower buds in both tomato and Arabidopsis. To characterize the specific regions of the RIP1 promoter that might be essential for its maximum activity and specificity in fruits, we made stable transgenic lines of tomato and Arabidopsis with 5'-deletion constructs. Characterization of these transgenic plants showed that the full length promoter is essential for its function. Overall, we report the identification and characterization of a ripening-induced promoter of tomato, which would be useful for the controlled manipulation of the ripening-related agronomic traits in genetic manipulation studies in future.

Lobule separator prosthesis to prevent adhesion of reconstructed ear lobe.

An adhesion is a band of scar tissue that binds two parts of the tissue together, which develops when the body's repair mechanisms respond to any tissue disturbance, such as surgery, infection, trauma, or radiation. Prevention of unwanted scar bands is of utmost importance to develop esthetic and healthy tissue. This article describes a technique to prevent the adhesion of the surgically reconstructed ear lobule with facial skin, using novel lobule separator prosthesis.

Functional and aesthetic rehabilitation of a geriatric patient with cleft palate: a case report.

Cleft palate defect with complete edentulism in elderly patients presents a prosthodontic challenge for complete denture fabrication. Such large defects are very difficult to restore by surgical intervention and have direct consequence on such functions as mastication and speech, on aesthetics, and on the patient's mental attitude. This article describes a case report in which a 76-year-old female patient underwent restoration with a closed hollow bulb obturator for a large cleft palate defect.

miR-135a targets IRS2 and regulates insulin signaling and glucose uptake in the diabetic gastrocnemius skeletal muscle.

Although aberrant miRNA signatures are associated with diabetes, yet, the status and role of altered miRNAs in the diabetic skeletal muscle is currently poorly understood. Here, we report that 41 miRNAs are altered in the diabetic gastrocnemius skeletal muscle and of these, miR-135a that is identified as a critical regulator of myogenesis, is significantly up-regulated. IRS2 is predicted as its potential putative target and its levels are down-regulated in the diabetic gastrocnemius skeletal muscle. In C2C12 cells, while miR-135a levels decreased during differentiation, IRS2 levels were up-regulated. miR-135a significantly reduced IRS2 protein levels and its 3'UTR luciferase reporter activity and these were blunted by the miR-135a inhibitor and mutation in the miR-135a binding site. Knock-down of endogenous miR-135a levels increased IRS2 at the mRNA and protein levels. miR-135a also attenuated insulin stimulated phosphorylation and activation of PI3Kp85α and Akt and glucose uptake. miR-135a levels were also found to be elevated in the human diabetic skeletal muscle. In-vivo silencing of miR-135a alleviated hyperglycemia, improved glucose tolerance and significantly restored the levels of IRS2 and p-Akt in the gastrocnemius skeletal muscle of db/db mice without any effect on their hepatic levels. These suggest that miR-135a targets IRS2 levels by binding to its 3'UTR and this interaction regulates skeletal muscle insulin signaling.

Three-dimensional orientation of iris in an ocular prosthesis using a customized scale.

The success of an ocular prosthesis depends largely on the correct orientation of the iris disk. Various methods have been put forth to achieve this. This article emphasizes one such simplified method, wherein a customized scale has been used to orient the iris disk mediolaterally, superoinferiorly, and anteroposteriorly in an ocular prosthesis. A scleral wax pattern was fabricated. The customized scale was used to measure the dimension and orientation of the natural iris. These measurements were then transferred to the scleral wax pattern with the customized scale. An iris disk was fabricated using black crayon on the scleral wax pattern according to the measurements. The scleral wax pattern, including the iris disk, was then placed in the eye socket to verify its dimension and orientation. A prefabricated iris disk was modified according to the measured dimensions and transferred to the final scleral wax pattern. The transfer of these dimensions to the definitive prosthesis was achieved successfully, ultimately improving the patient's social and psychological well being.

Mechanism of Ocular Penetration of Lipophilic Drugs from Lipophilic Vehicles.

Since eyedrops have conventionally been formulated in aqueous vehicles, ocular pharmacokinetic studies are generally performed using aqueous buffers to identify physicochemical properties of the drug and the vehicles that influence drug absorption. In recent years, biocompatible lipophilic vehicles are increasingly finding application in ocular drug delivery; however, the mechanism of drug penetration from these non-aqueous vehicles is poorly understood. This study aims to compare ocular penetration of the model lipophilic drug curcumin when incorporated into lipophilic vehicles. To elucidate whether intrinsic solubility in the lipophilic vehicle influences ocular penetration, a curcumin solution and suspension were prepared in medium chain triglycerides (MCT) and squalane, respectively. Ocular penetration and distribution of curcumin from both vehicles was compared and evaluated qualitatively and quantitatively ex vivo. Significantly greater and faster penetration was observed from the squalane suspension than from the MCT solution in all ocular tissues. Our results suggest that the ability of lipophilic drugs to partition out of lipophilic vehicles and into cell membranes, rather than their intrinsic solubility in the lipophilic vehicle, determines the rate and extent of their ocular penetration.

Tongues Tied by Orofacial Myofunctional Therapy about Tongue Tie: A Narrative Review.

Aim and Background: The respective review articles aim is to provide an overview as well as describes and enlists different orofacial myofunctional therapy exercises as a modality for tongue tie secondary to surgery.Tongue tie is the basically a connection that joints base of tongue to the floor of mouth. This leads to difficulties various difficulties such as altered speech, oral habits, maligned teeth and many more. During formative years, most children successfully treated of tongue tie by releasing it, but problems start after its correction. That it may can reappear or may lead to same difficulties as prior. Parents and clinicians are only concerned about speech and aesthetics after release of tongue tie. But OMT plays important role ore and post-surgical procedure. OMT help in proper tongue posture along with reducing the probability of tissue reattachment after surgery by exercises. This therapy positively influenced functions by reducing deleterious habits. Methods: A review of relevant literature is predicated on articles found using free text terms, mesh terms, and some basic tongue tie as well as tongue tie release pamphlets that were published in English up until the year 2023 in the electronic databases PubMed, EBSCO, Scopus, Google Scholar, and Web of Science. With the aid of mesh keywords, the initial search yielded 38-40 articles; 20-35 were chosen depending on the requirements. Also we searched for orofacial myofunctional exercises or exercises recommended after tongue tie release. Results: Various exercises enlisted in our article that will guide a individual before and after tongue tie release which will give positive outcomes such as proper tongue posture, speech, swallow, regained aesthetics and self-esteem. Conclusion: Tongue plays an important role in development of perioral structures as well as in the swallow to good speech articulation and dental occlusion. So, as pediatric dentist its important know that after release of tongue tie what to do and how to maintain. This review article is focused on the various orofacial myofunctional therapy techniques employed for tongue tie but not a single one to describe them. Clinical significance: Our pertaining review act as a guide for clinicians as well as individuals to manage tongue tie after its release. How to cite this article: Shah SS, Agarwal PV, Rathi N, et al. Tongues Tied by Orofacial Myofunctional Therapy about Tongue Tie: A Narrative Review. Int J Clin Pediatr Dent 2024;17(1):109-113.