Kidney development to kidney organoids and back again.

Kidney organoid technology has led to a renaissance in kidney developmental biology. The complex underpinnings of mammalian kidney development have provided a framework for the generation of kidney cells and tissues from human pluripotent stem cells. Termed kidney organoids, these 3-dimensional structures contain kidney-specific cell types distributed similarly to in vivo architecture. The adult human kidney forms from the reciprocal induction of two disparate tissues, the metanephric mesenchyme (MM) and ureteric bud (UB), to form nephrons and collecting ducts, respectively. Although nephrons and collecting ducts are derived from the intermediate mesoderm (IM), their development deviates in time and space to impart distinctive inductive signaling for which separate differentiation protocols are required. Here we summarize the directed differentiation protocols which generate nephron kidney organoids and collecting duct kidney organoids, making note of similarities as much as differences. We discuss limitations of these present approaches and discuss future directions to improve kidney organoid technology, including a greater understanding of anterior IM and its derivatives to enable an improved differentiation protocol to collecting duct organoids for which historic and future developmental biology studies will be instrumental.

Exploration of the Sclerotinia sclerotiorum-Brassica pathosystem: advances and perspectives in omics studies.

The polyphagous phytopathogen Sclerotinia sclerotiorum causing Stem rot disease is a major biotic stress in Brassica, and affects the yield and quality in various crops of agricultural significance. It affects the crop at pre-maturity which causes a reduction in the seed yield and deteriorates the oil quality in rapeseeds and Indian mustard globally. The hemibiotrophic nature and long persistence in the soil as sclerotia have made this pathogen difficult to manage through conventional agronomical practices. Hence, for alternative strategies, it is important to understand the basic aspects of the pathogen and the pathogenesis processes in the host. The current developments in technologies for omics studies including whole-genomes, transcriptomes, proteomes, and metabolomes have deciphered various genes, transcription factors, effectors and their target molecules involved in interaction, disease establishment and pathogen progress in the host tissues. The current review encompasses the studies that were conducted to decipher the Brassica-S. sclerotiorum pathosystem and the molecular factors identified through multi-omics studies for their application in building resistance to Sclerotinia stem rot disease in the susceptible cultivars of oilseed Brassica.

Functional maturation of kidney organoid tubules: PIEZO1-mediated Ca2+ signaling.

Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.

Flow-enhanced vascularization and maturation of kidney organoids in vitro.

Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

Strategies for utilization of crop wild relatives in plant breeding programs.

Crop wild relatives (CWRs) are weedy and wild relatives of the domesticated and cultivated crops, which usually occur and are maintained in natural forms in their centres of origin. These include the ancestors or progenitors of all cultivated species and comprise rich sources of diversity for many important traits useful in plant breeding. CWRs can play an important role in broadening genetic bases and introgression of economical traits into crops, but their direct use by breeders for varietal improvement program is usually not advantageous due to the presence of crossing or chromosome introgression barriers with cultivated species as well as their high frequencies of agronomically undesirable alleles. Linkage drag may subsequently result in unfavourable traits in the subsequent progeny when segments of the genome linked with quantitative trait loci (QTL), or a phenotype, are introgressed from wild germplasm. Here, we first present an overview in regards to the contribution that wild species have made to improve biotic, abiotic stress tolerances and yield-related traits in crop varieties, and secondly summarise the various challenges which are experienced in interspecific hybridization along with their probable solutions. We subsequently suggest techniques for readily harnessing these wild relatives for fast and effective introgression of exotic alleles in pre-breeding research programs.

Genetic variability, combining ability and molecular diversity-based parental line selection for heterosis breeding in field corn (Zea mays L.).

BACKGROUND: The demand of maize crop is increasing day by day, hence to reduce the production and demand gap, there is a need to extract the high yielding parental lines to improve per se yield of the hybrids, which could help to enhance the productivity in maize crops. METHODS AND RESULTS: The present investigation was carried out to select the best medium maturing inbred lines, among a set of 118 inbred lines. Based on the Duncan multiple range test, out of 118 lines, 16 inbred lines were selected on the basis of its high yield per se and flowering time. The molecular diversity was carried out using SSR markers linked to heterotic QTL and up on diversity analysis it classified selected genotypes in to three distinct groups. Among the selected inbred lines, a wider genetic variability and molecular diversity were observed. A total of 39 test crosses were generated after classifying 16 inbred lines in to three testers and thirteen lines (based on per se grain yield and molecular diversity) and crossing them in line × tester manner. CONCLUSION: Combining ability analysis of these parental lines showed that female parents, PML 109, PML 110, PML 111, PML 114 and PML 116 showed additive effect for KRN and grain yield, whereas male parents, PML 46, and PML 93 showed epistatic effect for KRN and PML 102 showed epistatic effect for grain yield. The generated information in the present investigation may be exploited for heterosis breeding in filed corn. KEY MESSAGES: To tackle the balanced dietary requirement of Indian population; we focused to enhance the productivity of maize hybrids using genetically broad based, elite, diverse inbred lines. Combination of selection criterion, not only augment the productivity but also improves the quality of hybrid/s.

Interleukin-1ß Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis.

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined.Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo, in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids.Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro, stimulation of purified human kidney SCs and human kidney organoids with IL-1ß recapitulated the molecular events observed in vivo, inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1ß stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury.Conclusions Our findings define a connection between IL-1ß and metabolic switch in fibrosis initiation and progression and highlight IL-1ß and MYC as potential therapeutic targets in tubulointerstitial diseases.

Bioavailability, Efficacy and Safety of Generic Immunosuppressive Drugs for Kidney Transplantation: A Systematic Review and Meta-Analysis.

BACKGROUND: Concerns exist over the extrapolation of bioavailability studies of generic immunosuppressive drugs in healthy volunteers, regarding their efficacy and safety in kidney transplant recipients. We conducted a meta-analysis of trials examining the bioavailability of generic (test) immunosuppressive drugs relative to their brand (reference) counterparts in healthy volunteers, based on the US Food and Drug Administration requirements for approval of generics, and their efficacy and safety in kidney transplant recipients. METHODS: Eligible studies were identified in PubMed, Cochrane Central Register of Controlled Trials, Scopus, ClinicalTrials.gov, and conference abstracts. RESULTS: Twenty crossover trials of healthy volunteers (n = 641) and 6 parallel-arm randomized controlled trials of kidney transplant recipients (n = 594) were identified. The 90% CI of the pooled test-to-reference drug ratio for maximum or peak plasma concentration (Cmax) and area under the plasma concentration time-curve from time 0 to time of last determinable concentration (AUC(0-t)) fell within the required range (0.80-1.25) for cyclosporine (Cmax 0.91; 90% CI 0.86-0.95; and AUC(0-t) 0.97; 90% CI 0.94-1.00), tacrolimus (Cmax 1.17; 90% CI 1.09-1.24; and AUC(0-t) 1.00; 90% CI 0.97-1.03) and mycophenolate mofetil (Cmax 0.98; 90% CI 0.96-1.01; and AUC(0-t) 1.00; 90% CI 0.99-1.01). In subgroup analyses, some generic cyclosporine formulations did not meet criteria for bioequivalence. No significant differences were observed in the time to maximum plasma concentration and terminal plasma half-life between generic and brand drugs. In parallel-arm trials, generic cyclosporine was non-inferior to brand counterpart in terms of acute allograft rejection, infections, and death. CONCLUSIONS: Not all generic immunosuppressive drugs have similar relative bioavailability to their brand name counterparts. Evidence on their efficacy and safety is inconclusive. Tighter regulatory requirement for approval of generic drugs with narrow therapeutic index is needed.

Outcomes of different concentrations of human amniotic fluid in a keratoconjunctivitis sicca-induced mouse model.

To compare the effects of different concentrations of topical human amniotic fluid (HAF) in a mouse model of dry eye, forty C57BL/6 mice were divided into 4 treatment groups: 20 % HAF, 50 % HAF, 100 % HAF, and isotonic salt solution (control). Dry eye was induced by an injection of botulinum toxin B into the lacrimal gland. Tear production, ocular surface fluorescein staining, and blink rate were evaluated in each mouse at 5 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Regarding tear production, 20, 50, and 100 % HAF groups were all different from the control group (P < 0.001) at week 1. However, there were no statistically significant differences between the 20, 50, and 100 % HAF groups. At week 2, 20, 50, and 100 % HAF groups had significant improvement in staining score and were significantly different from the control group (P = 0.047, P = 0.005, and P = 0.001, respectively). No difference in spontaneous blink rate was observed between groups, at any time point. Goblet cell density was significantly decreased in the control group compared to the HAF treatment groups. All tested concentrations of topical HAF were effective and superior than the control in this keratoconjunctivitis sicca-induced mouse model. Further studies are needed to evaluate the effects of HAF on the human ocular surface.

Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G.

BACKGROUND: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. METHOD: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. RESULT: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase-3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. CONCLUSION: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells.

Protective effects of lipoic acid on chrysene-induced toxicity on Müller cells in vitro.

PURPOSE: This study evaluates the toxic effects of chrysene (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro and investigates whether the inhibitor lipoic acid can reverse the chrysene-induced toxic effects. METHODS: MIO-M1 cells were exposed to varying concentrations of chrysene with or without lipoic acid. Cell viability was measured by a trypan blue dye exclusion assay. Caspase-3/7 activity was measured by a fluorochrome assay. Lactate dehydrogenase (LDH) release was quantified by an LDH assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with a 2',7'-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay. Intracellular ATP content was determined by the ATPLite kit. RESULTS: MIO-M1 cells showed significantly decreased cell viability, increased caspase-3/7 activity, LDH release at the highest chrysene concentration, elevated ROS/RNS levels, decreased ΔΨm value, and decreased intracellular ATP content after exposure to 300, 500, and 1,000 µM chrysene compared with the control. Pretreatment with 80 µM lipoic acid reversed loss of cell viability in 500-µM-chrysene-treated cultures (24.7%, p<0.001). Similarly, pretreatment with 80 µM lipoic acid before chrysene resulted in decreased caspase-3/7 activities (75.7%, p<0.001), decreased ROS/RNS levels (80.02%, p<0.001), increased ΔΨm values (86%, p<0.001), and increased ATP levels (40.5%, p<0.001) compared to 500-µM-chrysene-treated cultures. CONCLUSIONS: Chrysene, a component of cigarette smoke, can diminish cell viability in MIO-M1 cells in vitro by apoptosis at the lower concentrations of Chrysene (300 and 500 µM) and necrosis at the highest concentration. Moreover, mitochondrial function was particularly altered. However, lipoic acid can partially reverse the cytotoxic effect of chrysene. Lipoic acid administration may reduce or prevent Müller cell degeneration in retinal degenerative disorders.

ATP/ADP biosensor organoids for drug nephrotoxicity assessment.

Drug nephrotoxicity is a common healthcare problem in hospitalized patients and a major limitation during drug development. Multi-segmented kidney organoids derived from human pluripotent stem cells may complement traditional cell culture and animal experiments for nephrotoxicity assessment. Here we evaluate the capability of kidney organoids to investigate drug toxicity in vitro. Kidney organoids express renal drug transporters, OAT1, OAT3, and OCT2, while a human proximal tubular cell line shows the absence of OAT1 and OAT3. Tenofovir and aristolochic acid (AA) induce proximal tubular injury in organoids which is ameliorated by an OAT inhibitor, probenecid, without damage to podocytes. Similarly, cisplatin causes proximal tubular damage that can be relieved by an OCT inhibitor, cimetidine, collectively suggesting the presence of functional OATs and OCTs in organoid proximal tubules. Puromycin aminonucleoside (PAN) induced segment-specific injury in glomerular podocytes in kidney organoids in the absence of tubular injury. Reporter organoids were generated with an ATP/ADP biosensor, which may be applicable to high-throughput screening in the future. In conclusion, the kidney organoid is a useful tool for toxicity assessment in the multicellular context and may contribute to nephrotoxicity assessment during drug development.

Designed and validated novel allele-specific primer to differentiate Kernel Row Number (KRN) in tropical field corn.

BACKGROUND: Kernel row number (KRN) is an important yield component trait with a direct impact on the productivity of maize. The variability in KRN is influenced by the inflorescence meristem size, which is determined by the CLAVATA-WUSCHEL pathway. A CLAVATA receptor-like protein, encoded by the FASCIATED EAR2 (fea2gene), enhances the growth of inflorescence meristem and is thus involved in the determination of KRN. The amplicon sequencing-based method was employed to dissect the allelic variation of the fea2 gene in tropical field corn. METHODOLOGY/PRINCIPAL FINDING: Amplicon-based sequencing of AI 535 (Low KRN) and AI 536 (High KRN) was undertaken for the gene fea 2 gene that codes for KRN in maize. Upon multiple sequence alignment of both sequences, A to T transversion at the 1311 position was noticed between Low KRN and High KRN genotypes resulting in different allelic forms of a fea2 gene in tropical maize. An allele-specific primer 1311 fea2.1 was designed and validated that can differentiate High and Low KRN genotypes. CONCLUSION/SIGNIFICANCE: Maize has high variability for KRN and is exemplified by the wide values ranging from 8-26 KRN in the maize germpalsm. The sequence-based approach of SNP detection through the use of a specific primer facilitated the detection of variation present in the target trait. This makes it possible to capture these variations in the early generation. In the study, the PCR-based differentiation method described for the identification of desirable high KRN genotypes would augment the breeding programs for improving the productivity of field corn.

Indexing Resilience to Heat and Drought Stress in the Wild Relatives of Rapeseed-Mustard.

Wild species are weedy relatives and progenitors of cultivated crops, usually maintained in their centres of origin. They are rich sources of diversity as they possess many agriculturally important traits. In this study, we analysed 25 wild species and 5 U triangle species of Brassica for their potential tolerance against heat and drought stress during germination and in order to examine the early seedling stage. We identified the germplasms based on the mean membership function value (MFV), which was calculated from the tolerance index of shoot length, root length, and biochemical analysis. The study revealed that B. napus (GSC-6) could withstand high temperatures and drought. Other genotypes that were tolerant to the impact of heat stress were B. tournefortii (RBT 2002), D. gomez-campoi, B. tournefortii (Rawa), L. sativum, and B. carinata (PC-6). C. sativa resisted drought but did not perform well when subjected to high temperatures. Tolerance to drought was observed in B. fruticulosa (Spain), B. tournefortii (RBT 2003), C. bursa-pastoris (late), D. muralis, C. abyssinica (EC694145), C. abyssinica (EC400058) and B. juncea (Pusa Jaikisan). This investigation contributes to germplasm characterization and the identification of the potential source of abiotic stress tolerance in the Brassica breeding programme. These identified genotypes can be potential sources for transferring the gene(s)/genomic regions that determine tolerance to the elite cultivars.

Metabolomic profiling and its association with the bio-efficacy of Aspergillus niger strain against Fusarium wilt of guava.

Bio-control agents are the best alternative to chemicals for the successful management of plant diseases. The fungus Aspergillus niger is known to produce diverse metabolites with antifungal activity, attracting researchers to exploit it as a bio-control agent for plant disease control. In the present study, 11 A. niger strains were isolated and screened for their antagonism against the guava wilt pathogen under in vitro and in planta conditions. Strains were identified morphologically and molecularly by sequencing the internal transcribed spacer (ITS), ß-tubulin, and calmodulin genes. The strains were evaluated through dual culture, volatile, and non-volatile methods under an in vitro study. AN-11, AN-6, and AN-2 inhibited the test pathogen Fusarium oxysporum f. sp. psidii (FOP) at 67.16%, 64.01%, and 60.48%, respectively. An in planta study was conducted under greenhouse conditions with 6 months old air-layered guava plants (var. Allahabad Safeda) by pre- and post-inoculation of FOP. The AN-11 strain was found to be effective under both pre- and post-inoculation trials. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis was carried out to characterize the volatile compounds of the most potential strain, A. niger. The hexane soluble fraction showed the appearance of characteristic peaks of hexadecenoic acid methyl ester (4.41%), 10-octadecanoic acid methyl ester (3.79%), dodecane (3.21%), undecane (3.19%), gibepyrone A (0.15%), 3-methylundecane (0.36%), and citroflex A (0.38%). The ethyl acetate fraction of the bio-control fungi revealed the occurrence of major antifungal compounds, such as acetic acid ethyl ester (17.32%), benzopyron-4-ol (12.17%), 1,2,6-hexanetriol (7.16%), 2-propenoic acid ethanediyl ester (2.95%), 1-(3-ethyloxiranyl)-ethenone (0.98%), 6-acetyl-8-methoxy dimethyl chromene (0.96%), 4-hexyl-2,5-dihydro dioxo furan acetic acid (0.19%), and octadecanoic acid (1.11%). Furthermore, bio-control abilities could be due to hyper-parasitism, the production of secondary metabolites, and competition for sites and nutrients. Indeed, the results will enrich the existing knowledge of metabolomic information and support perspectives on the bio-control mechanism of A. niger.

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

Upstream sequence of fatty acyl-CoA reductase (FAR6) of Arabidopsis thaliana drives wound-inducible and stem-specific expression.

An Arabidopsis mutant line T90, exhibiting a stem-specific and wound-responsive GUS expression was identified from a population of Arabidopsis thaliana tagged with a promoterless ß-glucuronidase (GUS) in the T-DNA. Sequence flanking the insertion from the right border was amplified by TAIL PCR and cloned. The insertion was located in the third chromosome, 57 bp upstream of the ATG start codon in 5' untranslated region (UTR) of the fatty acyl-CoA reductase 6 (FAR6) gene. RT-PCR analysis of the FAR6 gene revealed that the gene is expressed predominantly in stem tissue. Semi-quantitative RT-PCR showed that the expression is also induced by wounding in the epidermal layer of mature stem internodes. The transcription initiation site (TSS) was identified by 5' RACE PCR. Different 5' deletion fragments of the promoter sequences were developed and linked to the GUS reporter gene as transcriptional fusions and the expression patterns of GUS were histochemically analyzed in transgenic Arabidopsis plants. Sequences from -510 bp upstream to the transcriptional start site were sufficient to exhibit wound-inducible GUS expression in the stems. The addition of further upstream sequences (-510 to -958, -1,400 or -1,456) enhanced and extended the wound-inducible GUS expression throughout the mature stem.

Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids.

Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity of these organoid structures. Transcriptomic analyses provide insight into maturation and transporter activities that represent kidney functions. However, functional assays in organoids are necessary to demonstrate the activity of these transport proteins in live tissues. The three-dimensional (3D) architecture adds complexity to real-time assays in kidney organoids. Here, we develop a functional assay using live imaging to assess transepithelial transport of rhodamine 123 (Rh123), a fluorescent substrate of P-glycoprotein (P-gp), in organoids affixed to coverslip culture plates for accurate real-time observation. The identity of organoid structures was probed using Lotus Tetragonolobus Lectin (LTL), which binds to glycoproteins present on the surface of proximal tubules. Within 20 min of the addition of Rh123 to culture media, Rh123 accumulated in the tubular lumen of organoids. Basolateral-to-apical accumulation of the dye/marker was reduced by pharmacologic inhibition of MDR1 or OCT2, and OCT2 inhibition reduced the Rh123 uptake. The magnitude of Rh123 transport was maturation-dependent, consistent with MDR1 expression levels assessed by RNA-seq and immunohistochemistry. Specifically, organoids on day 21 exhibit less accumulation of Rh123 in the lumen unlike later-stage organoids from day 30 of differentiation. Our work establishes a live functional assessment in 3D kidney organoids, enabling the functional phenotyping of organoids in health and disease.

Draft genome sequencing and secretome profiling of Sclerotinia sclerotiorum revealed effector repertoire diversity and allied broad-host range necrotrophy.

White mold commonly known as Sclerotinia sclerotiorum causes stem rot disease and has emerged as one of the major fungal pathogens of oilseed Brassica across the world. In the present study, consistently virulent S. sclerotiorum isolate "ESR-01" was sequenced and an assembly size of ~ 41 Mb with 328 scaffolds having N50 of 447,128 was obtained. Additionally, 27,450 single nucleotide polymorphisms (SNPs) were identified from 155 scaffolds against S. sclerotiorum 1980 isolate, with an average SNP density of ~ 1.5 per kb genome. 667 repetitive elements were identified and approximately comprised 7% of the total annotated genes. The DDE_1 with 454 in numbers was found to be the most abundant and accounts for 68% of the total predicted repetitive elements. In total, 3844 simple sequence repeats are identified in the 328 scaffolds. A total of 9469 protein-coding genes were predicted from the whole genome assembly with an average gene length of 1587 bp and their distribution as 230.95 genes per Mb in the genome. Out of 9469 predicted protein-coding genes, 529 genes were observed encoding the CAZymes (Carbohydrate-Active enzymes) capable of degradation of the complex polysaccharides. Glycosyltransferase (GT) families were most abundant (49.71%) among the predicted CAZymes and GT2 (23%), GT4 (20%), and glycoside hydrolase (GH) 23% with GH18 (11%) were the prominent cell wall degrading enzyme families in the ESR-01 secretome. Besides this, 156 genes essential for the pathogen-host interactions were also identified. The effector analysis in the whole genome proteomics dataset revealed a total of 57 effector candidates (ECs) and 27 of them were having their analogs whereas the remaining 30 were novel ones. Eleven selected ECs were validated experimentally by analyzing the expression profile of the ESR-01 isolate of S. sclerotiorum. Together, the present investigation offers a better understanding of the S. sclerotiorum genome, secretome, and its effector repertoire which will help in refining the present knowledge on S. sclerotiorum-Brassica interactions and necrotrophic lifestyle of the phytopathogen in general.

Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery.

Organoids serve as a novel tool for disease modeling in three-dimensional multicellular contexts. Static organoids, however, lack the requisite biophysical microenvironment such as fluid flow, limiting their ability to faithfully recapitulate disease pathology. Here, we unite organoids with organ-on-a-chip technology to unravel disease pathology and develop therapies for autosomal recessive polycystic kidney disease. PKHD1-mutant organoids-on-a-chip are subjected to flow that induces clinically relevant phenotypes of distal nephron dilatation. Transcriptomics discover 229 signal pathways that are not identified by static models. Mechanosensing molecules, RAC1 and FOS, are identified as potential therapeutic targets and validated by patient kidney samples. On the basis of this insight, we tested two U.S. Food and Drug Administration-approved and one investigational new drugs that target RAC1 and FOS in our organoid-on-a-chip model, which suppressed cyst formation. Our observations highlight the vast potential of organoid-on-a-chip models to elucidate complex disease mechanisms for therapeutic testing and discovery.