Synthesis of unsymmetric phosphorotrithioates by sequential coupling of 1,1-dichloro-N,N-diethylphosphanamine with thiols and sulfenyl chloride.

Herein, we present the first, one-step, direct synthesis of unsymmetric phosphorotrithioates through a process involving sequential coupling of 1,1-dichloro-N,N-diethylphosphanamine with thiols and sulfenyl chloride. This method showcases excellent functional group tolerance, substrate compatibility, and mild reaction conditions, offering a streamlined approach for the challenging phosphorotrithioate synthesis. Additionally, the applicability of this method can be extended to the synthesis of mixed phosphoroselenodithioates.

Synthesis of mixed phosphorotrithioates via thiol coupling with bis(diisopropylamino)chlorophosphine and sulphenyl chloride.

In this study, we report a novel and efficient one-pot synthesis of mixed phosphorotrithioates under mild conditions at ambient temperature, obviating the requirement for supplementary additives. The method's versatility stems from its utilization of diverse thiols as nucleophilic reactants, 1-chloro-N,N,N',N'-tetraisopropylphosphanediamine [bis(diisopropylamino)chlorophosphine] as the phosphorus precursor, and various sulphenyl chlorides as sources of electrophilic sulfur. Notably, our investigation extends beyond mixed phosphorotrithioates to encompass the synthesis of phosphoroselenodithioates, underscoring the broad applicability of this synthetic protocol.

BF3-Et2O promoted bifunctionalization of aldehydes for the synthesis of arylmethyl substituted organophosphorus compounds.

A simple and efficient protocol for the synthesis of arylmethyl substituted organophosphorus compounds is presented. This method involves the reaction of diphenyl phosphite with aldehydes in the presence of BF3-Et2O. In this method, BF3-Et2O plays a dual role, as it facilitates the generation of both hydrophosphonylated intermediate and phenol from diphenyl phosphite. A significant feature of this approach is its tolerance to the presence of external nucleophiles, such as phenol, aliphatic thiols, indole and 3-methylanisole. The simplicity of the reaction conditions and the high yields achieved make this method promising for applications in areas where phosphonate compounds are of interest.

BF3-Et2O Promoted Heteronucleophilic Addition Reactions for the Synthesis of Unsymmetrical gem-Diarylmethyl Thioethers.

Herein, we present the BF3/BF4- mediated activation of diphenyl phosphite for the formation of thionium ions from aldehydes and thiophenols. These reactive species subsequently undergo reaction with in situ generated phenol, resulting in the synthesis of diarylmethyl thioethers. It was demonstrated that the addition of external phenol in the reaction produced unsymmetrical gem-diarylmethyl thioethers in good yields.

Association of vitamin D deficiency and vitamin D receptor (VDR) gene single-nucleotide polymorphism (rs7975232) with risk of preeclampsia.

BACKGROUND: Preeclampsia has a multifactorial-yet-elusive etiology. Recent reports suggest a link between preeclampsia and vitamin D (VD) metabolic axis. Genetic variations like single-nucleotide polymorphisms (SNPs) of vitamin D receptor (VDR) gene can alter the metabolic role of VD, which have been shown by several genetic association studies. However, there is discordance among these studies. OBJECTIVE: The current study aimed to investigate the association of VDR gene polymorphism (ApaI) and VD deficiency with risk of developing preeclampsia. PATIENTS AND METHOD: In this case-control study, 40 preeclamptic and 40 normotensive pregnant women were compared for VD status and VDR gene polymorphism. Serum 25-hydroxyvitamin-D [25(OH) D] level was determined by enzyme-linked immunosorbent assay (ELISA) and VDR gene polymorphism Apa1 was analyzed by Allele specific polymerase chain reaction (AS-PCR) using sequence specific primers. RESULTS: Serum levels of 25(OH) D were very low but comparable in both preeclamptic and normotensive pregnant women. The difference between the two groups were not statistically significant (p = .423). VDR gene polymorphism ApaI (rs7975232) was found not to have significant association with the risk of developing preeclampsia. The frequencies of wild genotype (GG) in preeclamptic and normotensive women were 27.5% and 22.5% respectively. A total of 25% of preeclamptic women had mutant homozygous genotype (TT) and 17.5% of normotensive women had mutant homozygous genotype. The frequency of mutant heterozygous genotype (GT) in preeclamptic patients was 47.5% and in normotensive women was 60%. The variation of wild and mutant genotypes between the two groups was not statistically significant (p > .05). CONCLUSION: This study showed that VDR gene polymorphism (ApaI) and VD deficiency are not associated with the risk of preeclampsia.

Evaluation of Renessans (Iodine Complex Molecule) Safety in Human Beings: An Open-Labeled Clinical Study.

Extensive studies on evaluation of effectiveness/toxicity of different oral doses of iodine have not been explored yet. An open-labeled phase I clinical studies were conducted using iodine complex based research compound called Renessans. Study groups were observed for development of any adverse/serious adverse events and alteration in laboratory values of vital organs, TSH and T4 hormones before and after the administration of the products. Out of 31 consented individuals, 24 healthy individuals participated in the study. Rate of occurrence of mild Adverse Events (AEs) in group A was 8.3% while in Group B it was 33.33% but these Adverse Events were self-resolving. After completion of study treatment blood serum iodine was reported to 3522.88 µg/l while mean urine iodine concentration (MUIC) was greater than 2000 µg/l. Hormonal and vital organ's testing revealed that all parameters of TSH and T4, LFT, CBC, RFT remained unaltered except from ALT-SGPT (P-value = .006) and AST-SGOT (P-value = .02). From all of these findings, it can be inferred that the use of Renessans formulations did not pose any sort of risk to human body and can be considered safe through this pilot study.

Tf2O- and Cu(OTf)2-Assisted Acylamination Reaction of Unactivated Alcohols with Nitriles: A One-Pot P(IV) Activation, Stereoretention in Cycloalkanols and Deprotection Approach.

Described herein is a simple, novel, one-pot acylamination reaction of unactivated alcohols. This reaction employs the combination of PCl3 and triflic anhydride (Tf2O) or copper triflate Cu(OTf)2, which serves as a source of P(IV)-activated complex for nitriles to react under the Ritter-type mechanism. The synthetic utility of Tf2O-promoted reactions was demonstrated by its effectiveness to generate different acylaminated products. By employing Cu(OTf)2, this method represents a rare example of α-selective acylamination reaction. With chiral cycloalkanols, using the Cu(OTf)2-promoted procedure, acylaminated products are formed with complete retention of configuration. The synthetic utility of the copper-assisted reaction in acetonitrile was readily demonstrated as a mild deprotection strategy.

Reference equations for evaluation of spirometry function tests in South Asia, and among South Asians living in other countries.

BACKGROUND: There are few data to support accurate interpretation of spirometry data in South Asia, a major global region with a high reported burden of chronic respiratory disease. METHOD: We measured lung function in 7453 healthy men and women aged ≥18 years, from Bangladesh, North India, South India, Pakistan and Sri Lanka, as part of the South Asia Biobank study. First, we assessed the accuracy of existing equations for predicting normal forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and FEV1/FVC ratio. Then, we used our data to derive (n=5589) and internally validate (n=1864) new prediction equations among South Asians, with further external validation among 339 healthy South Asians living in Singapore. RESULTS: The Global Lung Initiative (GLI) and National Health and Nutrition Examination Survey consistently overestimated expiratory volumes (best fit GLI-African American, mean±sd z-score: FEV1 -0.94±1.05, FVC -0.91±1.10; n=7453). Age, height and weight were strong predictors of lung function in our participants (p<0.001), and sex-specific reference equations using these three variables were highly accurate in both internal validation (z-scores: FEV1 0.03±0.99, FVC 0.04±0.97, FEV1/FVC -0.03±0.99) and external validation (z-scores: FEV1 0.31±0.99, FVC 0.24±0.97, FEV1/FVC 0.16±0.91). Further adjustment for study regions improves the model fit, with highest accuracy for estimation of region-specific lung function in South Asia. CONCLUSION: We present improved equations for predicting lung function in South Asians. These offer the opportunity to enhance diagnosis and management of acute and chronic lung diseases in this major global population.

Modified W-type configuration for a single-phase reduced parts count 81-level inverter.

The technology of modern power systems is revolutionizing as renewable energy sources are being integrated with electric power grids. In the form of inverters, power electronic converters are becoming an integral part of power systems due to their massive demand for grid integration of photovoltaic (PV) systems. Existing multilevel inverter topologies either require an output filter to get a sinusoidal voltage or generate a higher number of output voltage levels at the expense of many hardware resources. This paper presents a new single-phase 81-level inverter configuration with the name given as 'Modified W-Type Multilevel Inverter'. The proposed inverter configuration uses only eighteen power switches and four DC voltage sources to generate an 81-level output voltage approaching a sinusoidal waveform without an output filter. The general design equations are developed to calculate the number of switches, the number of voltage levels, and the number of DC sources for the proposed configuration. Loss and efficiency analysis is carried out that verifies a good practical efficiency of the proposed inverter configuration during the dynamic operation. A comparative analysis with the existing MLI topologies is also carried out that validates the effectiveness and novelty in reducing parts count and higher number of voltage levels. The proposed topology offers 1.04% total harmonic distortion of the output voltage which is within the benchmarks specified for grid integration without any filter requirements. The proposed inverter configuration is simulated in MATLAB/Simulink, and the results are validated by the design and development of a hardware prototype.

Transgene CpNAC68 from Wintersweet (Chimonanthus praecox) Improves Arabidopsis Survival of Multiple Abiotic Stresses.

The NAC (NAM, ATAFs, CUC) family of transcription factors (TFs) play a pivotal role in regulating all processes of the growth and development of plants, as well as responses to biotic and abiotic stresses. Yet, the functions of NACs from non-model plant species remains largely uncharacterized. Here, we characterized the stress-responsive effects of a NAC gene isolated from wintersweet, an ornamental woody plant that blooms in winter when temperatures are low. CpNAC68 is clustered in the NAM subfamily. Subcellular localization and transcriptional activity assays demonstrated a nuclear protein that has transcription activator activities. qRT-PCR analyses revealed that CpNAC68 was ubiquitously expressed in old flowers and leaves. Additionally, the expression of CpNAC68 is induced by disparate abiotic stresses and hormone treatments, including drought, heat, cold, salinity, GA, JA, and SA. Ectopic overexpression of CpNAC68 in Arabidopsis thaliana enhanced the tolerance of transgenic plants to cold, heat, salinity, and osmotic stress, yet had no effect on growth and development. The survival rate and chlorophyll amounts following stress treatments were significantly higher than wild type Arabidopsis, and were accompanied by lower electrolyte leakage and malondialdehyde (MDA) amounts. In conclusion, our study demonstrates that CpNAC68 can be used as a tool to enhance plant tolerance to multiple stresses, suggesting a role in abiotic stress tolerance in wintersweet.

Involvement of ABA Responsive SVB Genes in the Regulation of Trichome Formation in Arabidopsis.

Trichome formation in Arabidopsis is regulated by several key regulators, and plants hormones such as gibberellin, salicylic acid, jasmonic acid and cytokinins have been shown to regulate trichome formation by affecting the transcription or activities of the key regulators. We report here the identification of two abscisic acid (ABA) responsive genes, SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) and SVB2 as trichome formation regulator genes in Arabidopsis. The expression levels of SVB and SVB2 were increased in response to ABA treatment, their expression levels were reduced in the ABA biosynthesis mutant aba1-5, and they have similar expression pattern. In addition to the trichome defects reported previously for the svb single mutant, we found that even though the trichome numbers were largely unaffected in both the svb and svb2 single mutants generate by using CRISPR/Cas9 gene editing, the trichome numbers were greatly reduced in the svb svb2 double mutants. On the other hand, trichome numbers were increased in SVB or SVB2 overexpression plants. RT-PCR results show that the expression of the trichome formation key regulator gene ENHANCER OF GLABRA3 (EGL3) was affected in the svb svb2 double mutants. Our results suggest that SVB and SVB2 are ABA responsive genes, and SVB and SVB2 function redundantly to regulate trichome formation in Arabidopsis.

PIP2, An Auxin Induced Plant Peptide Hormone Regulates Root and Hypocotyl Elongation in Arabidopsis.

Auxin is one of the traditional plant hormones, whereas peptide hormones are peptides with hormone activities. Both auxin and plant peptide hormones regulate multiple aspects of plant growth and development, and there are cross-talks between auxin and plant peptide hormones. PAMP-INDUCED SECRETED PEPTIDES (PIPs) and PIP-LIKEs (PIPLs) are a new family of plant peptide hormone, and PIPL3/TARGET OF LBD SIXTEEN 2 (TOLS2) has been shown to regulate lateral root formation in Arabidopsis. We report here the identification of PIP2 as an auxin response gene, and we found it plays a role in regulating root and hypocotyl development in Arabidopsis. By using quantitative RT-PCR, we found that the expression of PIP2 but not PIP1 and PIP3 was induced by auxin, and auxin induced expression of PIP2 was reduced in nph4-1 and arf19-4, the lost-of-function mutants of Auxin Response Factor 7 (ARF7) and ARF19, respectively. By generating and characterizing overexpressing transgenic lines and gene edited mutants for PIP2, we found that root length in the PIP2 overexpression plant seedlings was slightly shorter when compared with that in the Col wild type plants, but root length of the pip2 mutant seedlings remained largely unchanged. For comparison, we also generated overexpressing transgenic lines and gene edited mutants for PIP3, as well as pip2 pip3 double mutants. Surprisingly, we found that root length in the PIP3 overexpression plant seedlings is shorter than that of the PIP2 overexpression plant seedlings, and the pip3 mutant seedlings also produced short roots. However, root length in the pip2 pip3 double mutant seedlings is largely similar to that in the pip3 single mutant seedlings. On the other hand, hypocotyl elongation assays indicate that only the 35S:PIP2 transgenic plant seedlings produced longer hypocotyls when compared with the Col wild type seedlings. Further analysis indicates that PIP2 promotes cell division as well as cell elongation in hypocotyls. Taken together, our results suggest that PIP2 is an auxin response gene, and PIP2 plays a role in regulating root and hypocotyl elongation in Arabidopsis likely via regulating cell division and cell elongation.

Knockout of the entire family of AITR genes in Arabidopsis leads to enhanced drought and salinity tolerance without fitness costs.

BACKGORUND: Environmental stresses including abiotic stresses and biotic stresses limit yield of plants. Stress-tolerant breeding is an efficient way to improve plant yield under stress conditions. Genome editing by CRISPR/Cas9 can be used in molecular breeding to improve agronomic traits in crops, but in most cases, with fitness costs. The plant hormone ABA regulates plant responses to abiotic stresses via signaling transduction. We previously identified AITRs as a family of novel transcription factors that play a role in regulating plant responses to ABA and abiotic stresses. We found that abiotic stress tolerance was increased in the single, double and triple aitr mutants. However, it is unclear if the increased abiotic stress tolerance in the mutants may have fitness costs. RESULTS: We report here the characterization of AITRs as suitable candidate genes for CRISPR/Cas9 editing to improve plant stress tolerance. By using CRISPR/Cas9 to target AITR3 and AITR4 simultaneously in the aitr256 triple and aitr1256 quadruple mutants respectively, we generated Cas9-free aitr23456 quintuple and aitr123456 sextuple mutants. We found that reduced sensitivities to ABA and enhanced tolerance to drought and salt were observed in these mutants. Most importantly, plant growth and development was not affected even in the aitr123456 sextuple mutants, in whom the entire AITR family genes have been knocked out, and the aitr123456 sextuple mutants also showed a wild type response to the pathogen infection. CONCLUSIONS: Our results suggest that knockout of the AITR family genes in Arabidopsis enhanced abiotic stress tolerance without fitness costs. Considering that knock-out a few AITRs will lead to enhanced abiotic stress tolerance, that AITRs are widely distributed in angiosperms with multiple encoding genes, AITRs may be targeted for molecular breeding to improve abiotic stress tolerance in plants including crops.

Nanoparticle-based genetic transformation of Cannabis sativa.

Cannabis sativa (Cannabis) is a multipurpose plant species consisting of specific lineages that for centuries has either been artificially selected for the production of fiber or the psychoactive drug Δ9-tetrahydrocannabinol (THC). With the recent lifting of previous legal restrictions on consuming Cannabis, there has been a resurgence of interest in understanding and manipulating Cannabis genetics to enhance its compositions. Yet, recently developed approaches are not amenable to high-throughput gene stacking to study multi-genic traits. Here, we demonstrate an efficient nanoparticle-based transient gene transformation protocol where multiple gene plasmids can be expressed simultaneously in intact Cannabis leaf cells in a very short time (5 days). Constructs encoding two soybean transcription factors were co-grafted onto poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold nanoparticles (PEI-Au@SiO2). Infiltration of the DNA-PEI-Au@SiO2 into Cannabis leaf tissues resulted in the transcription of both soybean genes and the localization of fluorescent-tagged transcription factor proteins in the nuclei of Cannabis leaf cells including the trichomes, which are the cell types that biosynthesize valuable cannabinoid and terpene metabolites. Our study exemplifies a rapid transient gene transformation approach that will be useful to study the effects of gene stacking in Cannabis.

Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis.

The CRISPR/Cas9 genome editing technique has been widely used to generate transgene-free mutants in different plant species. Several different methods including fluorescence marker-assisted visual screen of transgene-free mutants and programmed self-elimination of CRISPR/Cas9 construct have been used to increase the efficiency of genome edited transgene-free mutant isolation, but the overall time length required to obtain transgene-free mutants has remained unchanged in these methods. We report here a method for fast generation and easy identification of transgene-free mutants in Arabidopsis. By generating and using a single FT expression cassette-containing CRISPR/Cas9 construct, we targeted two sites of the AITR1 gene. We obtained many early bolting plants in T1 generation, and found that about two thirds of these plants have detectable mutations. We then analyzed T2 generations of two representative lines of genome edited early bolting T1 plants, and identified plants without early bolting phenotype, i.e., transgene-free plants, for both lines. Further more, aitr1 homozygous mutants were successful obtained for both lines from these transgene-free plants. Taken together, these results suggest that the method described here enables fast generation, and at the mean time, easy identification of transgene-free mutants in plants.

Genome Editing to Integrate Seed Size and Abiotic Stress Tolerance Traits in Arabidopsis Reveals a Role for DPA4 and SOD7 in the Regulation of Inflorescence Architecture.

Both seed size and abiotic stress tolerance are important agronomic traits in crops. In Arabidopsis, two closely related transcription repressors DPA4 (Development-Related PcG Target in the APEX4)/NGAL3 and SOD7 (Suppressor of da1-1)/NGAL2 (NGATHA-like protein) function redundantly to regulate seed size, which was increased in the dpa4 sod7 double mutants. Whereas ABA-induced transcription repressors (AITRs) are involved in the regulation of ABA signaling and abiotic stress tolerance, Arabidopsis aitr2 aitr5 aitr6 (aitr256) triple mutant showed enhanced tolerance to drought and salt. Here we performed CRISPR/Cas9 genome editing to disrupt DPA4 and SOD7 in aitr256 mutant, trying to integrate seed size and abiotic stress tolerance traits in Arabidopsis, and also to examine whether DPA4 and SOD7 may regulate other aspects of plant growth and development. Indeed, seed size was increased in the dpa4 sod7 aitr256 quintuple mutants, and enhanced tolerance to drought was observed in the mutants. In addition, we found that shoot branching was affected in the dpa4 sod7 aitr256 mutants. The mutant plants failed to produce secondary branches, and flowers/siliques were distributed irregularly on the main stems of the plants. Floral organ number and fertility were also affected in the dpa4 sod7 aitr256 mutant plants. To examine if these phenotypes were dependent on loss-of-function of AITRs, dpa4 sod7 double mutants were generated in Col wild type background, and we found that the dpa4 sod7 mutant plants showed a phenotype similar to the dpa4 sod7 aitr256 quintuple mutants. Taken together, our results indicate that the integration of seed size and abiotic stress tolerance traits by CRISPR/Cas9 editing was successful, and our results also revealed a role of DPA4 and SOD7 in the regulation of inflorescence architecture in Arabidopsis.

GCN5 modulates trichome initiation in Arabidopsis by manipulating histone acetylation of core trichome initiation regulator genes.

KEY MESSAGE: Histone acetyltransferase GCN5 affects trichome initiation via mediating the expression of some core trichome initiation regulator genes in Arabidopsis. GENERAL CONTROL NON-REPRESSED PROTEIN5 (GCN5), a histone acetyltransferase involved in the regulation of cell differentiation, organ development, secondary metabolism, and plant responses to abiotic stresses, has recently been shown to modulate trichome branching in Arabidopsis. Here, we provide evidence that GCN5 is also involved in the regulation of trichome initiation. We found that mutation of GCN5 led to increased leaf trichome density in Arabidopsis. Quantitative RT-PCR results showed that the expression of CPC, GL1, GL2, and GL3, four well-known core trichome initiation regulator genes, was decreased in the gcn5 mutants. ChIP assays indicated that these four trichome initiation regulator genes are direct targets of GCN5. Consistent with these results, GCN5-mediated H3K14/K9 acetylation levels on the TSS regions of these genes were decreased. On the other hand, leaf trichome density was reduced in plants overexpressing GCN5, and both the transcript levels and GCN5-binding enrichments of CPC, GL1, GL2, and GL3 genes were elevated. Taken together, these data suggests that GCN5 affects trichome initiation by modulating the transcription activities of trichome initiation regulator genes via H3K9/14 acetylation.

Role Of Choledochoduodenostomy Revisited In The Era Of Minimal Invasive Procedures.

BACKGROUND: Present era of endoscopic and laparoscopic approaches have revolutionized surgical management of Common Bile Duct (CBD) stones. Open procedures like Choledochoduodenostomy (CDD) have become a rarity but are not completely obsolete. It may be considered a relatively safe alternative when dealing with recurrent, too large or impacted stones, a failed ERCP, and CBD stricture with stones. The aim of this study was to establish safety, efficiency, cost effectiveness and easy availability of CDD in selected patients. METHODS: In this observational study, the outcome of 90 consecutive patients undergoing CDD between 1st January 1995 and 31st Dec, 2016 in surgical unit C, Ayub Medical Complex, Abbottabad was reviewed. Choledochoduodenostomy was offered to patients who refused or had a failed ERCP and when CBD size was more than 1cm. Common Bile Duct was anastomosed to Duodenum using standard technique. RESULTS: Medical records of 90 patients (age range 34-96 years) were reviewed; 5 were excluded and 85 were included in the study. Complication rate was 31.76% (n=27) including respiratory complications in 16.47% (n=14), wound infection in 10.6% (n=9), anastomotic leak in 2.35% (n=2) and Cholangitis in 1.18% (n=1) and Mortality in 1.18% (n=1). There was no evidence of Sump Syndrome. Total cost of procedure was Rs.50 as admission fee, which included daily provision of food and there were no room charges. CONCLUSIONS: Choledochoduodenostomy is a safe procedure, with fewer complications and significantly low cost. In case of ERCP failure, CDD is recommended.