Household secondary attack rate amongst the susceptible contacts of COVID-19 cases and its epidemiological profile: A retrospective study from central Gujarat, India.

Introduction: Secondary attack rate (SAR) is a proportion of primary contacts developing the diseases within the incubation period upon exposure to a primary case among the total susceptible household contacts. The epidemiological profile and SAR will help in understanding the transmission dynamics of COVID-19 for further strengthening preventive and effective control measures. Objectives of the Study: The study was conducted with the primary objective to estimate the household SAR of COVID-19 cases at Mahisagar District, Gujarat, and to study the epidemiological profile of primary and secondary cases of COVID-19. Methodology: A retrospective study was conducted to estimate SAR among 245 confirmed cases of COVID-19 and 898 susceptible household contacts in Mahisagar district through telephonic interview and questionnaire by the Investigator team. Results: The household SAR was calculated from the current study in Mahisagar district, Gujarat, and was 13.9%. Among primary cases, 74.7% males were affected, and among secondary cases, 52.8% females were affected. The SAR among elderly was 23.9%, and the SAR among children was 3.9%. 74.1% household contacts had developed disease among symptomatic contacts as compared to asymptomatic household contacts. Conclusion: The household SAR in Mahisagar district, Gujarat, was found to be 13.9%. In primary cases, more males, and in secondary cases, more females were found to be affected. The household SAR was increased in elderly as compared to the younger age group. The SAR was more among the contacts of symptomatic cases than asymptomatic cases. Overall hospitalization in public hospitals was more than that in private hospitals.

Spectroscopic and quantum chemical investigations to explore the effect of intermolecular interactions in a diuretic drug: Hydrochlorothiazide.

Hydrochlorothiazide (HCTZ) being a diuretic drug widely used in anti-hypertensive therapy as it lowers the blood pressure by reducing the reabsorption of electrolytes in kidney resulting an increment of urine output and lowering the blood pressure. The purpose of the present work is to study the structural, vibrational and chemical properties of HCTZ based on its monomeric, dimeric and trimeric models by utilizing computational methods and experimental techniques. Density functional theory (DFT) with functional B3LYP and 6-311++G (d, p) basis set was used for a detailed computational study. Monomeric, dimeric and trimeric models of HCTZ have been studied for a better understanding of inter- and intramolecular hydrogen bonding. FT-IR (400-3800 cm-1) and FT-Raman (100-3600 cm-1) spectroscopy have been utilized for the characterization of HCTZ. The shifting in wavenumber of NH2 and OSO group were observed in dimer and trimer due to the formation of intermolecular hydrogen bonding. Quantum theory of atoms in molecules (QTAIM) along with natural bond orbital (NBO) analysis were performed to examine the nature and strength of hydrogen bonding which showed that all the interactions were medium and partially covalent in nature; transition from LP(3)O15 â†’ σ*(H46 â†’ N32) and LP(3)O39 â†’ σ*(H74 â†’ N51) were responsible for the formation of O15•••H46 and O39•••H74 H-bonds, respectively. HOMO-LUMO energies predicted the chemical reactivity and stability of the molecule and the energy gap for dimer (4.6240 eV) and trimer (4.0493 eV) was found to be lesser than the monomer (5.0888 eV) which showed that the dimer and trimer have predicted more chemical reactivity in comparison to monomer. The most electronegative electrostatic potential was observed around the OSO group and the most electropositive potential around the amide group from MEPS analysis. Global as well as local reactivity descriptors have predicted the reactivity and hence, stability of the title molecule.

A Study on Epidemiology of Unintentional Childhood Injury at One of the Districts of Gujarat State: An Indian Council of Medical Research Task Force Study.

Context: Childhood injuries are a less explored area of public health. In the presence of public health problems such as communicable and noncommunicable diseases, childhood injury has gained less attention from the health system. Aims: The aim of the study was to assess the prevalence of various types of unintentional injuries (UI) amongst children and to assess the factors associated with same. Settings and Design: The study was conducted at Ahmedabad District of Gujarat State, and this is a cross-sectional sStudy. Subjects and Methods: The current study was a part of a multicentric national level research supported by Indian Council of Medical Research. Total 11 sites were selected across India, which all followed a uniform methodology. One of the selected sites was Dholka taluka of Ahmedabad district, Gujarat. This cross-sectional study was conducted in 2341 households. The study participants were children aged from 6 months up to 18 years. A total of 3018 children and their caretakers were interviewed using software-based questionnaire. Statistical Analysis Used: Statistical analysis was performed by frequencies and Percentage, Chi-square Test, Z-Test. Results: The overall prevalence of injuries was 7.62%. Gender and area of residence were significantly associated with UI. Fall-related injuries had the highest prevalence (3.38%), followed by road traffic injuries (RTI) (1.62%). The prevalence of burn-related injuries and poisoning was higher among females compared to males. No mortality was observed due to UI. Conclusions: Majority of the injuries were of minor or trivial type. Fall-related injury and RTI were the most common types of UIs among children.

Cytochrome P450 Binding and Bioactivation of Tumor-Targeted Duocarmycin Agents.

Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

The Dolaflexin-based Antibody-Drug Conjugate XMT-1536 Targets the Solid Tumor Lineage Antigen SLC34A2/NaPi2b.

Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.

Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

Isomorphism: 'molecular similarity to crystal structure similarity' in multicomponent forms of analgesic drugs tolfenamic and mefenamic acid.

The non-steroidal anti-inflammatory drugs mefenamic acid (MFA) and tolfenamic acid (TFA) have a close resemblance in their molecular scaffold, whereby a methyl group in MFA is substituted by a chloro group in TFA. The present study demonstrates the isomorphous nature of these compounds in a series of their multicomponent solids. Furthermore, the unique nature of MFA and TFA has been demonstrated while excavating their alternate solid forms in that, by varying the drug (MFA or TFA) to coformer [4-di-methyl-amino-pyridine (DMAP)] stoichiometric ratio, both drugs have produced three different types of multicomponent crystals, viz. salt (1:1; API to coformer ratio), salt hydrate (1:1:1) and cocrystal salt (2:1). Interestingly, as anticipated from the close similarity of TFA and MFA structures, these multicomponent solids have shown an isomorphous relation. A thorough characterization and structural investigation of the new multicomponent forms of MFA and TFA revealed their similarity in terms of space group and structural packing with isomorphic nature among the pairs. Herein, the experimental results are generalized in a broader perspective for predictably identifying any possible new forms of comparable compounds by mapping their crystal structure landscapes. The utility of such an approach is evident from the identification of polymorph VI of TFA from hetero-seeding with isomorphous MFA form I from acetone-methanol (1:1) solution. That aside, a pseudopolymorph of TFA with di-methyl-formamide (DMF) was obtained, which also has some structural similarity to that of the solvate MFA:DMF. These new isostructural pairs are discussed in the context of solid form screening using structural landscape similarity.

Nitrofurantoin-melamine monohydrate (cocrystal hydrate): Probing the role of H-bonding on the structure and properties using quantum chemical calculations and vibrational spectroscopy.

Cocrystal monohydrate of nitrofurantoin (NF) with melamine (MELA) has been studied as NF is an antibacterial drug used for the treatment of urinary tract infections. The structure of nitrofurantoin-melamine-monohydrate (NF-MELA-H2O) is characterized by FT-IR and FT-Raman spectroscopy. The energies and vibrational frequencies of the optimized structures calculated using quantum chemical calculations. Supported by normal coordinate analyses and potential energy distributions (PEDs), the complete vibrational assignments recommended for the observed fundamentals of cocrystal hydrate. With the aim of inclusion of all the H-bond interactions, dimer of NF-MELA-H2O has been studied as only two molecules of cocrystal hydrate are present in the unit cell. By the study of dimeric model consistent assignment of the FT-IR and FT-Raman spectrum obtained. H-bonds are of essential importance in an extensive range of molecular sciences. The vibrational analyses depict existence of H-bonding (O-H⋯N) between water O-H and pyridyl N atom of MELA in both monomer and dimer. To probe the strength and nature of H-bonding in monomer and dimer, topological parameters such as electron density (ρBCP), Laplacian of electron density (∇2ρBCP), total electron energy density (HBCP) and H-bond energy (EHB) at bond critical points (BCP) are evaluated by quantum theory of atoms in molecules (QTAIM). Natural bond orbitals (NBOs) analyses are carried out to study especially the intra and intermolecular H-bonding and their second order stabilization energy (E(2)). The value of HOMO-LUMO energy band gap for NF-MELA-H2O (monomer and dimer both) is less than NF, showing more chemical reactivity for NF-MELA-H2O. Chemical reactivity has been described with the assistance of electronic descriptors. Global electrophilicity index (ω = 7.3992 eV) shows that NF-MELA-H2O behaves as a strong electrophile than NF. The local reactivity descriptors analyses such as Fukui functions, local softnesses and electrophilicity indices performed to determine the reactive sites within NF-MELA-H2O. In MEP map of NF-MELA (monomer and dimer) electronegative regions are about NO2 and C=O group of NF, although the electropositive regions are around NH2, N-H group and H2O molecule. Molar refractivity (MR) value of NF-MELA-H2O (monomer and dimer) lies within the range set by Lipinski's modified rules. This study could set as an example to study the H-bond interactions in pharmaceutical cocrystals.

Quality of life among elderly population residing in urban field practice area of a tertiary care institute of Ahmedabad city, Gujarat.

INTRODUCTION: Overall improvement in the living standards of country's population is leading to longer life expectancy. To emphasize the medical and psychological difficulties faced by geriatric people is essential to know status of their quality of life (QOL). METHODOLOGY: A community-based cross-sectional study was carried out at urban field practice area of one of the teaching institutes of Ahmedabad, Gujarat. Considering the prevalence of about 7.5% of 60 years and above people sample size of 250 was calculated. A predesigned questionnaire related to the QOL of elderly people devised by the World Health Organization-QOL was used. RESULTS: Mean age of the study population was 65.8 years with standard deviation of 5 years. Almost two-thirds of geriatrics were currently married and having spouse alive. List of common morbidities observed among study population was joint pain (42.8%), cataract (32.8%), hypertension (22.4%), diabetes mellitus (17.2%), and dental problems (12.4%). Scoring of QOL profile revealed that none of the geriatric had poor QOL, whereas 56% fall into category "good" and 50.8% had "excellent" QOL. QOL as per four different domains was significantly better among males as compared to females. Physical, environmental, and psychological domains were better in those who were educated and married individuals living with their spouse. CONCLUSION: Overall QOL was good to excellent. Social characteristics, such as education, marital status, and gender, all play role for the perceived QOL among the respondents.

Prevalence and microscopic studies of Sarcocystis infection in naturally infected water buffaloes (Bubalus bubalis) of Andhra Pradesh.

Sarcocystis species are the obligate tissue protozoan parasites of livestock causing clinical and subclinical disease resulting in downgrading of the meat and their products thereby leading to economic losses. The present study reveals the prevalence and distribution of sarcocystosis in water buffaloes (Bubalus bubalis) slaughtered at local abattoirs in A.P for a period of 1 year from June 2014 to May 2015. A total of 137 buffalo carcasses were screened grossly and microscopically organ wise viz., esophagus, tongue, heart, diaphragm and intercostal muscles. Out of 137 screened, 91 were infected with an overall prevalence of 66.42 %. Age wise analysis of 89 young male calves aged about 18-36 months old revealed 65.16 % (58/89) and 48 old she buffaloes (5-8 years) showed 68.75 % (33/48). The organ wise prevalence was highest in esophagus (51.82 %) followed by tongue (47.44 %), heart (29.92 %), diaphragmatic muscles (28.46 %) and intercostal muscles (18.24 %), respectively. Morphometric studies revealed the presence of two Sarcocystis species, i.e., S.levinei and S.fusiformis infection with a prevalence of 43.79 and 22.62 %, respectively, along with mixed infection rate of 43.06 %. Microscopic studies of S.levinei showed sarcocyst length/width/cyst wall thickness ranged between 0.31-0.69/0.09-0.12 mm/<1 µm, respectively, and bradyzoites with an average of 6.25 µm length/2.5 µm width. Similarly, S.fusiformis cyst ranged between 2 and 8.5 mm/1-3 mm/2-5 µm and bradyzoites with an average of 10 µm length/2.5 µm width. The histopathological studies revealed congestion and degenerative changes of myocytes along with infiltration of mononuclear cells.

Thermodynamic Investigation of Carbamazepine-Saccharin Co-Crystal Polymorphs.

Polymorphism in active pharmaceutical ingredients can be regarded as critical for the potential that crystal form can have on the quality, efficacy, and safety of the final drug product. The current contribution aims to characterize thermodynamic interrelationship of a dimorphic co-crystal, FI and FII, involving carbamazepine (CBZ) and saccharin (SAC) molecules. Supramolecular synthesis of CBZ-SAC FI and FII has been performed using thermokinetic methods and systematically characterized by differential scanning calorimetry, powder X-ray diffraction, solubility, and slurry measurements. According to the heat of fusion rule by Burger and Ramberger, FI (ΔHfus = 121.1 J/g; melting point, 172.5°C) and FII (ΔHfus = 110.3 J/g; melting point, 164.7°C) are monotropically related. The solubility and van't Hoff plot results suggest FI stable and FII metastable forms. This study reveals that CBZ-SAC co-crystal phases, FI or FII, could be stable to heat-induced stresses; however, FII converts to FI during solution-mediated transformation.

In vitro evaluation of acaricidal activity of novel green silver nanoparticles against deltamethrin resistance Rhipicephalus (Boophilus) microplus.

An investigation was undertaken to study, for the first time, in vitro acaricidal activity of green silver nanoparticles on deltamethrin resistance Rhipicephalus (Boophilus) microplus. The compounds tested were neem coated silver nanoparticles (N-Ag NPs), deltamethrin neem coated silver nanoparticles (DN-Ag NPs), 2, 3 dehydrosalannol (2,3 DHS), 2, 3 DHS coated silver nanoparticles (2, 3-DHS-Ag NPs), Quercetin dihydrate (QDH) and QDH coated silver nanoparticles (QDH-Ag NPs). Also included in this study, for the purpose of comparison, were neem leaf extract (NLE), silver nitrate (AgNO3) and deltamethrin (D). Acaricidal activity on larvae and adults of R. (B.) microplus was tested by larval packet test (LPT) and adult immersion test (AIT) respectively. In the LPT, 100% mortality was obtained at concentrations (ppm) of 360, 6000, 260, 200, 50, 300, 85, 600 and 200 for the compounds, D, NLE, Ag NO3, N-Ag NPs, DN-Ag NPs, 2, 3 DHS, 2, 3 DHS-Ag NPs, QDH, QDH-Ag NPs respectively. In AIT, the proportions of mortality and oviposition inhibition were proportionate but the reproductive index was inversely proportional to the concentration of the compounds used. The effect of DN-Ag NPs on mortality was the highest (93.33%) at 50ppm concentration. The mean reproductive index (0.01) and oviposition inhibition (99.16%) values were statistically significant when compared to control group. DN-Ag NPs showed significantly (P<0.05) lower LC50 (3.87ppm; 21.95ppm) and LC99 (53.05ppm; 90.06ppm) values against both the larvae and adults of R. (B.) microplus. The oviposition inhibiting ability of various compounds was determined to assess the reproductive performance of adult female ticks. The DN-Ag NPs had potent oviposition inhibitory activity with significantly lower IC50 and IC99 values compared to the rest of the treatments at 0.034 and 51.07ppm respectively. These results showed that the DN-Ag NPs had significant acaricidal activity against R. (B.) microplus.

First report of Cobboldia elephantis (Cobbold, 1866) larvae in a free ranging wild elephant from Andhra Pradesh, India.

Larvae of Cobboldia elephantis have been reported from the stomach of a free ranging wild elephant (Elephas maximus) while conducting post mortem examination at Palamner forest range, Chittoor district of Andhra Pradesh state, India. This is the first report of C. elephantis in free ranging wild elephant in Andhra Pradesh state, India.

Babesia bigemina infection in a 14-day old Jersey crossbred calf: a case report.

Babesia bigemina infection was diagnosed in a 14-day old Jersey female calf. The infected calf showed clinical symptoms of high fever, increased respiratory rate, pale conjunctival mucous membrane and haemoglobinuria. Blood smears were prepared and subjected for Giemsas staining method. Microscopic examination of the stained blood smear confirmed the characteristic intra-erythrocytic B. bigemina organisms.

Identification of candidate genes associated with positive and negative heterosis in rice.

To identify the genes responsible for yield related traits, and heterosis, massively parallel signature sequencing (MPSS) libraries were constructed from leaves, roots and meristem tissues from the two parents, 'Nipponbare' and '93-11', and their F1 hybrid. From the MPSS libraries, 1-3 million signatures were obtained. Using cluster analysis, commonly and specifically expressed genes in the parents and their F1 hybrid were identified. To understand heterosis in the F1 hybrid, the differentially expressed genes in the F1 hybrid were mapped to yield related quantitative trait loci (QTL) regions using a linkage map constructed from 131 polymorphic simple sequence repeat markers with 266 recombinant inbred lines derived from a cross between Nipponbare and 93-11. QTLs were identified for yield related traits including days to heading, plant height, plant type, number of tillers, main panicle length, number of primary branches per main panicle, number of kernels per main panicle, total kernel weight per main panicle, 1000 grain weight and total grain yield per plant. Seventy one QTLs for these traits were mapped, of which 3 QTLs were novel. Many highly expressed chromatin-related genes in the F1 hybrid encoding histone demethylases, histone deacetylases, argonaute-like proteins and polycomb proteins were located in these yield QTL regions. A total of 336 highly expressed transcription factor (TF) genes belonging to 50 TF families were identified in the yield QTL intervals. These findings provide the starting genomic materials to elucidate the molecular basis of yield related traits and heterosis in rice.

Planar Hall magnetoresistive aptasensor for thrombin detection.

The use of aptamer-based assays is an emerging and attractive approach in disease research and clinical diagnostics. A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using a planar Hall magnetoresistive (PHR) sensor in cooperation with superparamagnetic labels. A PHR sensor has the great advantages of a high signal-to-noise ratio, a small offset voltage and linear response in the low-field region, allowing it to act as a high-resolution biosensor. In the system presented here, the sensor has an active area of 50 µm × 50 µm with a 10-nm gold layer deposited onto the sensor surface prior to the binding of thiolated DNA primary aptamer. A polydimethylsiloxane well of 600-µm radius and 1-mm height was prepared around the sensor surface to maintain the same specific area and volume for each sensor. The sensor response was traced in real time upon the addition of streptavidin-functionalized magnetic labels on the sensor. A linear response to the thrombin concentration in the range of 86 pM-8.6 µM and a lower detection limit down to 86 pM was achieved by the proposed present method with a sample volume consumption of 2 µl. The proposed aptasensor has a strong potential for application in clinical diagnosis.

Cross-coupling reaction between arylboronic acids and carboranyl iodides catalyzed by graphene oxide (GO)-supported Pd(0) recyclable nanoparticles for the synthesis of carboranylaryl ketones.

Well-dispersed palladium(0) nanoparticles with small and narrow size distributions were synthesized conveniently on a graphene oxide (GO) surface. The GO-supported nano-Pd(0) was found to be a highly efficient and recyclable catalyst for the carbonylative cross-coupling reaction between arylboronic acids and aryl and carboranyl iodides, respectively. Benzophenone and a series of carboranylaryl ketones, 1-R-2-[C(=O)Ar]-1,2-C2B10H10 (R = H, Me, Ph; Ar = C6H5, C6H4-4-OMe and C6H4-4-F), were synthesized and fully characterized. The catalyst was recyclable at least three times with sustained activity.

Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors.

BACKGROUND: Rice blast caused by the fungus Magnaporthe oryzae is an important disease in virtually every rice growing region of the world, which leads to significant annual decreases of grain quality and yield. To prevent disease, resistance genes in rice have been cloned and introduced into susceptible cultivars. However, introduced resistance can often be broken within few years of release, often due to mutation of cognate avirulence genes in fungal field populations. RESULTS: To better understand the pattern of mutation of M. oryzae field isolates under natural selection forces, we used a next generation sequencing approach to analyze the genomes of two field isolates FJ81278 and HN19311, as well as the transcriptome of FJ81278. By comparing the de novo genome assemblies of the two isolates against the finished reference strain 70-15, we identified extensive polymorphisms including unique genes, SNPs (single nucleotide polymorphism) and indels, structural variations, copy number variations, and loci under strong positive selection. The 1.75 MB of isolate-specific genome content carrying 118 novel genes from FJ81278, and 0.83 MB from HN19311 were also identified. By analyzing secreted proteins carrying polymorphisms, in total 256 candidate virulence effectors were found and 6 were chosen for functional characterization. CONCLUSIONS: We provide results from genome comparison analysis showing extensive genome variation, and generated a list of M. oryzae candidate virulence effectors for functional characterization.

Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.