Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase binding in 40S ribosome assembly.

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AFs) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate "handover" from one highly related AF to another, remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the binding of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1 into its functional site. Inactive Tsr3 blocks Rio1 function, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

Clinical value assessment for serum hsa_tsr013526 in the diagnosis of gastric carcinoma.

Gastric carcinoma (GC) is a malignant tumor that is detrimental to human health. Transfer RNA-derived small RNAs are a newly identified class of noncoding small RNAs with specific biological functions that are aberrantly expressed in cancer. The aim of this study was to investigate the potential of hsa_tsr013526 as a biomarker for GC. Quantitative real-time fluorescence polymerase chain reaction was used to detect the expression level of hsa_tsr013526. The molecular characteristics of hsa_tsr013526 were verified by agarose gel electrophoresis, Sanger sequencing, and separation of nuclear and cytoplasmic RNA fractions. By testing the receiver operating characteristic (ROC) curves, the diagnostic efficiency of GC using hsa_tsr013526 was determined. Finally, we predicted the downstream of hsa_tsr013526 using functional assays and bioinformatics analysis. Serum expression of hsa_tsr013526 was higher in GC patients than in healthy donors. Serum expression showed differential changes in GC patients, gastritis patients, and healthy donors. Chi-squared tests showed that high expression of hsa_tsr013526 was significantly correlated with T stage, lymphatic metastasis, and tumor node metastasis stage. ROC curve analysis indicated that GC patients could be discriminated from healthy donors or gastritis patients based on their serum levels of hsa_tsr013526. Furthermore, hsa_tsr013526 expression was significantly reduced in postoperative GC patients (p = .0016). High expression of hsa_tsr013526 promotes gastric cancer cell proliferation, invasion, and migration. Serum hsa_tsr013526 was stable and specific, and could be used for dynamic monitoring of GC patients. Therefore, hsa_tsr013526 may be a new biomarker for the diagnosis and postoperative monitoring of GC patients.

Node-negative colon cancer: histological, molecular, and stromal features predicting disease recurrence.

BACKGROUND: Within the group of node-negative colon cancer patients, presumed to have a good prognosis, a significant percentage of patients develops cancer-recurrence. Current high-risk features prove inadequate to select these particular high-risk patients. In the process of tailor-made care and shared decision-making the need to identify these patients grows. In this study we investigate the value of adding molecular markers and the tumour-stroma ratio (TSR) to conventional histological tumour staging methods to improve the selection of high risk patients. METHODS: We retrospectively analysed 201 patients diagnosed with TNM-stage I-II colon cancer and treated by complete oncological resection between November 1st 2002 and December 31st 2012 at the Jeroen Bosch Hospital. Conventional histological tumour staging, BRAF mutations, KRAS mutations, MSI status and TSR were determined. Differences between groups based on TSR and mutation status, in disease free survival were analysed using Cox-Regression analyses. RESULTS: Poorly differentiated histology (p = 0.002), high-TSR (p = 0.033), BRAF-mutation (p = 0.008) and MSI (p = 0.011) were identified as significant risk factors for cancer recurrence. The risk of recurrence increased in the presence of both a BRAF-mutation and high-TSR compared to the absence of both factors or presence of only one factor (HR = 3.66 BRAF-mt/TSR-low (p = 0.006), HR 2.82 BRAF-wt/TSR-high (p = 0.015), HR = 4.39 BRAF-mt/TSR-high (p = 0.023)). This was also seen in tumours with MSI and high-TSR (HR = 2.46 MSS/TSR-high (p = 0.041), HR = 3.31 MSI/TSR-high (p = 0.045). CONCLUSION: Judging by the higher HR for the combination of the prognostic factors TSR and BRAF compared to the HRs of these prognostic factors individually, the prognostication for disease free survival can be improved by determining both TSR and BRAF instead of BRAF alone, as is done in current daily practise. In this study MSI also shows additional value to TSR in the prognostication of disease free survival. Adopting TSR into daily diagnostics will be of additional value next to currently used molecular markers in risk stratification of patients with node negative colon cancer and is therefore advised.

Association Mapping of Resistance to Tan Spot in the Global Durum Panel.

Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is an important disease of durum and common wheat worldwide. Compared with common wheat, less is known about the genetics and molecular basis of tan spot resistance in durum wheat. We evaluated 510 durum lines from the Global Durum Wheat Panel (GDP) for sensitivity to the necrotrophic effectors (NEs) Ptr ToxA and Ptr ToxB and for reaction to Ptr isolates representing races 1 to 5. Overall, susceptible durum lines were most prevalent in South Asia, the Middle East, and North Africa. Genome-wide association analysis showed that the resistance locus Tsr7 was significantly associated with tan spot caused by races 2 and 3, but not races 1, 4, or 5. The NE sensitivity genes Tsc1 and Tsc2 were associated with susceptibility to Ptr ToxC- and Ptr ToxB-producing isolates, respectively, but Tsn1 was not associated with tan spot caused by Ptr ToxA-producing isolates, which further validates that the Tsn1-Ptr ToxA interaction does not play a significant role in tan spot development in durum. A unique locus on chromosome arm 2AS was associated with tan spot caused by race 4, a race once considered avirulent. A novel trait characterized by expanding chlorosis leading to increased disease severity caused by the Ptr ToxB-producing race 5 isolate DW5 was identified, and this trait was governed by a locus on chromosome 5B. We recommend that durum breeders select resistance alleles at the Tsr7, Tsc1, Tsc2, and the chromosome 2AS loci to obtain broad resistance to tan spot.

Assessment of ACC and P450 Genes Expression in Wild Oat (Avena ludoviciana) in Different Tissues Under Herbicide Application.

Target-site resistance (TSR) and non-target-site resistance (NTSR) to herbicides in arable weeds are increasing rapidly all over the world and threatening universal food safety. Resistance to herbicides that inhibit ACCase activity has been identified in wild oat. In this study, expression of ACC1, ACC2, CYP71R4 and CYP81B1 genes under herbicide stress conditions were studied in two TSR (resistant in the residue Ile1781-Leu and Ile2041-Asn of ACCase) biotypes, two NTSR biotypes and one susceptible biotype of A. ludoviciana for the first time. Treated and untreated biotypes with ACCase-inhibitor clodinafop propargyl herbicide were sampled from the stem and leaf tissues at 24 h after treatment. Our results showed an increase in gene expression levels in different tissues of both types of resistance biotypes that occurred under herbicide treatment compared with non-herbicide treatment. In all samples, the expression levels of leaf tissue in all studied genes were higher than in stem tissue. The results of ACC gene expression showed that the expression level of ACC1 was significantly higher than that of ACC2. Also, expression levels of TSR biotypes were higher than NTSR biotypes for the ACC1 gene. For both CYP71R4 and CYP81B1 genes, the expression ratio increased significantly in TSR and NTSR biotypes in different tissues after herbicide treatment. In contrast, the expression levels of CYP genes in NTSR biotypes were higher than in TSR biotypes. Our results support the hypothesis that the reaction of plants to herbicide is carried out through a different regulation of genes, which can be the result of the interaction of resistance type in the target or non-target-site.

Addition of an Fc-IgG induces receptor clustering and increases the in vitro efficacy and in vivo anti-tumor properties of the thrombospondin-1 type I repeats (3TSR) in a mouse model of advanced stage ovarian cancer.

OBJECTIVES: Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound. METHODS: Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated. RESULTS: Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer. CONCLUSION: The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer.

First Asp-2078-Gly Mutation Conferring Resistance to Different ACCase Inhibitors in a Polypogon fugax Population from China.

Asia minor bluegrass (Polypogon fugax) is a common and problematic weed throughout China. P. fugax that is often controlled by acetyl-CoA carboxylase (ACCase) inhibitors in canola fields. Herein, we confirmed a P. fugax population (R) showing resistance to all ACCase inhibitors tested with resistance indexes ranging from 5.4-18.4. We further investigated the resistance mechanisms of this R population. Molecular analyses revealed that an amino acid mutation (Asp-2078-Gly) was present in the R population by comparing ACCase gene sequences of the sensitive population (S). In addition, differences in susceptibility between the R and S population were unlikely to be related to herbicide metabolism. Furthermore, a new derived cleaved amplified polymorphic sequence (dCAPS) method was developed for detecting the Asp-2078-Gly mutation in P. fugax efficiently. We found that 93.75% of plants in the R population carried the Asp-2078-Gly mutation, and all the herbicide-resistant phenotype of this R population is inseparable from this mutation. This is the first report of cross resistance to ACCase inhibitors conferred by the Asp-2078-Gly target-site mutation in P. fugax. The research suggested the urgent need to improve the diversity of weed management practices to prevent the widespread evolution of herbicide resistance in P. fugax in China.

Dostarlimab an Inhibitor of PD-1/PD-L1: A New Paradigm for the Treatment of Cancer.

Immunomodulation checkpoints usually adopted by healthy cells by tumors might cause an imbalance between host surveillance and tumor progression. Several tumors are incredibly resistant to standard treatment. The dynamic and long-lasting tumor regressions caused by antibodies targeting the PD-1/PD-L1 checkpoint have suggested a rebalancing of the host-tumor relationship. Checkpoint antibody inhibitors, like anti-PD-1/PD-L1, are unique inhibitors that reduce tumor growth by modulating the interaction between immune cells and tumor cells. These checkpoint inhibitors are swiftly emerging as a highly promising strategy for treating cancer because they produce impressive antitumor responses while having a limited number of adverse effects. Over the past several years, numerous checkpoint antibody inhibitors pointing to PD-1, PDL-1, and CTLA-4 have been available on the market. Despite its enormous success and usefulness, the anti-PD treatment response is restricted to certain kinds of cancer. This restriction can be attributed to the inadequate and diverse PD-1 expression in the tumor (MET) micro-environment. Dostarlimab (TSR-042), a drug that interferes with the PD-1/PD-L1 pathway, eliminates a crucial inhibitory response of an immune system and, as a result, has the potential to cause severe or deadly immune-mediated adverse effects. As cancer immunotherapy, dostarlimab enhances the antitumor immune response of the body.

Intratumoral Budding in Pretreatment Biopsies, among Tumor Microenvironmental Components, Can Predict Prognosis and Neoadjuvant Therapy Response in Colorectal Adenocarcinoma.

Background and Objectives: The prediction of the prognosis and effect of neoadjuvant therapy is vital for patients with advanced or unresectable colorectal carcinoma (CRC). Materials and Methods: We investigated several tumor microenvironment factors, such as intratumoral budding (ITB), desmoplastic reaction (DR), and Klintrup-Mäkinen (KM) inflammation grade, and the tumor-stroma ratio (TSR) in pretreatment biopsy samples (PBSs) collected from patients with advanced or unresectable CRC. A total of 85 patients with 74 rectal carcinomas and 11 colon cancers treated at our hospital were enrolled; 66 patients had curative surgery and 19 patients received palliative treatment. Results: High-grade ITB was associated with recurrence (p = 0.002), death (p = 0.034), and cancer-specific death (p = 0.034). Immature DR was associated with a higher grade of clinical tumor-node-metastasis stage (cTNM) (p = 0.045), cN category (p = 0.045), and cM category (p = 0.046). The KM grade and TSR were not related to any clinicopathological factors. High-grade ITB had a significant relationship with tumor regression in patients who received curative surgery (p = 0.049). Conclusions: High-grade ITB in PBSs is a potential unfavorable prognostic factor for patients with advanced CRC. Immature DR, TSR, and KM grade could not predict prognosis or therapy response in PBSs.

O-Fucosylation of ADAMTSL2 is required for secretion and is impacted by geleophysic dysplasia-causing mutations.

ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-ß binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose-modified TSRs are subsequently elongated to a glucose ß1-3-fucose (GlcFuc) disaccharide by ß1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2-/- but not in B3GLCT-/- cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.

Oxygen limitation fails to explain upper chronic thermal limits and the temperature size rule in mayflies.

An inability to adequately meet tissue oxygen demands has been proposed as an important factor setting upper thermal limits in ectothermic invertebrates (especially aquatic species) as well as explaining the observed decline in adult size with increased rearing temperature during the immature stages (a phenomenon known as the temperature size rule, or TSR). We tested this by rearing three aquatic insects (the mayflies Neocloeon triangulifer and two species of the Cloeon dipterum complex) through their entire larval life under a range of temperature and oxygen concentrations. Hyperoxia did not extend upper thermal limits, nor did it prevent the loss of size or fertility experienced near upper chronic thermal limits. At moderate temperatures, the TSR pattern was observed under conditions of hyperoxia, normoxia and hypoxia, suggesting little or no influence of oxygen on this trend. However, for a given rearing temperature, adults were smaller and less fecund under hypoxia as a result of a lowering of growth rates. These mayflies greatly increased the size of their gills in response to lower dissolved oxygen concentrations but not under oxygen-saturated conditions over a temperature range yielding the classic TSR response. Using ommatidium diameter as a proxy for cell size, we found the classic TSR pattern observed under moderate temperature conditions was due primarily to a change in the number of cells rather than cell size. We conclude overall that a failure to meet tissue oxygen demands is not a viable hypothesis for explaining either the chronic thermal limit or TSR pattern in these species.

Dostarlimab in the treatment of recurrent or primary advanced endometrial cancer.

Immune checkpoint inhibitors have demonstrated significant clinical activity across various tumor subtypes; however, their utility in gynecologic malignancies has thus far proven modest. Since the identification of a molecular subclassification system for endometrial cancer (EC), research in immune checkpoint inhibitor therapies has been focusing on certain subgroups predictive for response, particularly microsatellite instability hypermutated/DNA mismatch repair-deficient subtype. Dostarlimab, a PD-1 inhibitor, has demonstrated preliminary evidence of clinical activity and acceptable safety profile in patients with across recurrent EC, particularly microsatellite instability-hypermutated/DNA mismatch repair-deficient EC. This review outlines existing data for the efficacy and safety of dostarlimab in recurrent or advanced-stage EC.

Selective retina therapy and thermal stimulation of the retina: different regenerative properties - implications for AMD therapy.

BACKGROUND: Selective Retina Therapy (SRT), a photodisruptive micropulsed laser modality that selectively destroys RPE cells followed by regeneration, and Thermal Stimulation of the Retina (TSR), a stimulative photothermal continuous wave laser modality that leads to an instant sublethal temperature increase in RPE cells, have shown therapeutic effects on Age-related Macular Degeneration (AMD) in mice. We investigate the differences between both laser modalities concerning RPE regeneration. METHODS: For PCR array, 6 eyes of murine AMD models, apolipoprotein E and nuclear factor erythroid-derived 2- like 2 knock out mice respectively, were treated by neuroretina-sparing TSR or SRT. Untreated litter mates were controls. Eyes were enucleated either 1 or 7 days after laser treatment. For morphological analysis, porcine RPE/choroid organ cultures underwent the same laser treatment and were examined by calcein vitality staining 1 h and 1, 3 or 5 days after irradiation. RESULTS: TSR did not induce the expression of cell-mediators connected to cell death. SRT induced necrosis associated cytokines as well as inflammation 1 but not 7 days after treatment. Morphologically, 1 h after TSR, there was no cell damage. One and 3 days after TSR, dense chromatin and cell destruction of single cells was seen. Five days after TSR, there were signs of migration and proliferation. In contrast, 1 h after SRT a defined necrotic area within the laser spot was seen. This lesion was closed over days by migration and proliferation of adjacent cells. CONCLUSIONS: SRT induces RPE cell death, followed by regeneration within a few days. It is accompanied by necrosis induced inflammation, RPE proliferation and migration. TSR does not induce immediate RPE cell death; however, migration and mitosis can be seen a few days after laser irradiation, not accompanied by necrosis-associated inflammation. Both might be a therapeutic option for the treatment of AMD.

Performance Enhancement for Full-Duplex Relaying with Time-Switching-Based SWIPT in Wireless Sensors Networks.

Full-duplex (FD) with simultaneous wireless information and power transfer (SWIPT) in wireless ad hoc networks has received increased attention as a technology for improving spectrum and energy efficiency. This paper studies the outage performance for a SWIPT-based decode-and-forward (DF) FD relaying network consisting of a single-antenna source S, a two-antenna relay R, and a multi-antenna destination D. Specifically, we propose four protocols, namely static time-switching factor with selection combining (STSF-SC), static time-switching factor with maximal ratio combining (STSF-MRC), optimal dynamic time-switching factor with selection combining (ODTSF-SC), and optimal dynamic time-switching factor with maximal ratio combining (ODTSF-MRC) to fully investigate the outage performance of the proposed system. In particular, the optimal time-switching factor from the ODTSF-SC and ODTSF-MRC methods is designed to maximize the total received data at the destination. In this context, we derive exact closed-formed expressions for all schemes in terms of the outage probability (OP). Finally, the Monte Carlo simulations are conducted to corroborate the theoretical analysis's correctness and the proposed schemes' effectiveness.

Studies of congenital cataract-related TSR1 mutation and its expression in the lens.

Congenital cataract (CC) is a rare disease with dysplasia of the lens, mainly characterized by partial or complete opacity of the lens. The molecular basis of the disease is complex, mutations in over 266 genes associated with congenital cataracts had been reported. In this study, a novel congenital cataract candidate gene TSR1 was identified by whole genome sequencing and Sanger sequencing in a Chinese congenital cataract family. The TSR1 c.202-1G>A substitution affected splicing of TSR1 mRNA was confirmed by a minigene assay. The expression of TSR1 in mouse lens, anterior lens capsule of age-related cataract patients and 24-week human fetal lens were determined by RT-PCR, Western blotting, and immunofluorescence assays. The expression of TSR1 in the embryonic and different developmental stages of the mouse lens was confirmed by analyzing the iSyTE database. The expression of TSR1 was down-regulated in the lens-specific CBP:p300 double knockout mouse, and a set of genes with the same expression pattern of Tsr1 in the CBP:p300 double knockout mouse lens were extracted for protein-protein interaction network analysis, and six proteins were screened for direct interaction with Tsr1. GO function analysis indicated that Tsr1 might play a role in the MAPK-Erk signaling pathway in addition to its involvement in ribosome assembly. This study provided valuable research clues to further clarify the function of Tsr1 in the lens.

Distinct domains in the matricellular protein Lonely heart are crucial for cardiac extracellular matrix formation and heart function in Drosophila.

The biomechanical properties of extracellular matrices (ECMs) are critical to many biological processes, including cell-cell communication and cell migration and function. The correct balance between stiffness and elasticity is essential to the function of numerous tissues, including blood vessels and the lymphatic system, and depends on ECM constituents (the "matrisome") and on their level of interconnection. However, despite its physiological relevance, the matrisome composition and organization remain poorly understood. Previously, we reported that the ADAMTS-like protein Lonely heart (Loh) is critical for recruiting the type IV collagen-like protein Pericardin to the cardiac ECM. Here, we utilized Drosophila as a simple and genetically amenable invertebrate model for studying Loh-mediated recruitment of tissue-specific ECM components such as Pericardin to the ECM. We focused on the functional relevance of distinct Loh domains to protein localization and Pericardin recruitment. Analysis of Loh deletion constructs revealed that one thrombospondin type 1 repeat (TSR1-1), which has an embedded WXXW motif, is critical for anchoring Loh to the ECM. Two other thrombospondin repeats, TSR1-2 and TSR1-4, the latter containing a CXXTCXXG motif, appeared to be dispensable for tethering Loh to the ECM but were crucial for proper interaction with and recruitment of Pericardin. Moreover, our results also suggested that Pericardin in the cardiac ECM primarily ensures the structural integrity of the heart, rather than increasing tissue flexibility. In conclusion, our work provides new insights into the roles of thrombospondin type 1 repeats and advances our understanding of cardiac ECM assembly and function.

A Plasmodium falciparum C-mannosyltransferase is dispensable for parasite asexual blood stage development.

C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (PfDPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of PfDPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a PfDPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. PfDPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that PfDPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions.

Oxygen Limitation at the Larval Stage and the Evolution of Maternal Investment per Offspring in Aquatic Environments.

Oxygen limitation and surface area to volume relationships of the egg were long thought to constrain egg size in aquatic environments, but more recent evidence indicates that egg size per se does not influence oxygen availability to embryos. Here, we suggest that investment per offspring is nevertheless constrained in aquatic anamniotes by virtue of oxygen transport in free-living larvae. Drawing on the well-supported assumption that oxygen limitation is relatively pronounced in aquatic versus terrestrial environments and that oxygen limitation is particularly severe in warm aquatic environments, we employ comparative methods in the Amphibia to investigate this problem. Across hundreds of species and two major amphibian clades, the slope of species mean egg diameter over habitat temperature is negative for species with aquatic larvae but is positive or neutral for species featuring terrestrial eggs and no larvae. Yet across species with aquatic larvae, the negative slope of egg diameter over temperature is similar whether eggs are laid terrestrially or aquatically, consistent with an oxygen constraint arising at the larval stage. Finally, egg size declines more strongly with temperature for species that cannot breathe aerially before metamorphosis compared with those that can. Our results suggest that oxygen transport in larvae (not eggs) constrains investment per offspring. This study further extends the generality of temperature-dependent oxygen limitation as a mechanism driving the temperature-size rule in aquatic systems.