Hydrated [3+2] Cyclotelomerization of Butafulvenes to Create Multiple Contiguous Fully Substituted Carbon Centers.

The construction of multiple continuous fully substituted carbon centers, which serve as unique structural motif in natural products, is a challenging topic in organic synthesis. Herein, we report a hydrated [3+2] cyclotelomerization of butafulvenes to create contiguous fully substituted carbon backbone. In the presence of scandium triflate, all-carbon skeleton with spiro fused tricyclic ring can be constructed in high diastereoselectivity by utilizing butafulvene as the synthon. Mechanistic studies suggest that this atom-economic reaction probably proceeds through a synergistic process containing butafulvenes dimerization and nucleophilic attack by water. In addition, the tricyclic product can undergo a series of synthetic derivatizations, which highlights the potential applications of this strategy. The recyclability of Sc(OTf)3 has also been demonstrated to show its robust performance in this hydrated cyclotelomerization.

Catalytic Undirected Meta-Selective C-H Borylation of Metallocenes.

Metallocenes are privileged backbones in the fields of synthetic chemistry, catalysis, polymer science, etc. Direct C-H functionalization is undoubtedly the simplest approach for tuning the properties of metallocenes. However, owing to the presence of multiple identical C(sp2 )-H sites, this protocol often suffers from low reactivity and selectivity issues, especially for the regioselective synthesis of 1,3-difunctionalized metallocenes. Herein, an efficient iridium-catalyzed meta-selective C-H borylation of metallocenes is reported. With no need of preinstalled directing groups, this approach enables a rapid synthesis of various boronic esters based on benzoferrocenes, ferrocenes, ruthenocene, and related half sandwich complex. A broad range of electron-deficient and -rich functional groups are all compatible with the process. Notably, C-H borylation of benzoferrocenes takes place exclusively at the benzene ring, which is likely ascribed to the shielding effect of pentamethylcyclopentadiene. The synthetic utility is further demonstrated by easy scalability to gram quantities, the conversion of boron to heteroatoms including N3 , SePh, and OAc, as well as diverse cross-coupling reactions.

Nickel-catalyzed divergent Mizoroki-Heck reaction of 1,3-dienes.

Developing efficient strategies to realize divergent arylation of dienes has been a long-standing synthetic challenge. Herein, a nickel catalyzed divergent Mizoroki-Heck reaction of 1,3-dienes has been demonstrated through the regulation of ligands and additives. In the presence of Mn/NEt3, the Mizoroki-Heck reaction of dienes delivers linear products under Ni(dppe)Cl2 catalysis in high regio- and stereoselectivities. With the help of catalytic amount of organoboron and NaF, the use of bulky ligand IPr diverts the selectivity from linear products to branched products. Highly aryl-substituted compounds can be transformed from dispersive Mizoroki-Heck products programmatically. Preliminary experimental studies are carried out to elucidate the role of additives.

Self-paced Multi-view Learning for CT-based severity assessment of COVID-19.

Prior studies for the task of severity assessment of COVID-19 (SA-COVID) usually suffer from domain-specific cognitive deficits. They mainly focus on visual cues based on single cognitive functions but fail to reconcile the valuable information from other alternative views. Inspired by the cognitive process of radiologists, this paper shifts naturally from single-symptom measurements to a multi-view analysis, and proposes a novel Self-paced Multi-view Learning (SPML) framework for automated SA-COVID. Specifically, the proposed SPML framework first comprehensively aggregates multi-view contexts in lung infection with different measure paradigms, i.e., Global Feature Branch, Texture Feature Branch, and Volume Feature Branch. In this way, multiple-perspective clues are taken into account to reflect the most essential pathological manifestation on CT images. To alleviate small-sample learning problems, we also introduce an optimization with self-paced learning strategy to cognitively increase the characterization capabilities of training samples by learning from simple to complex. In contrast to traditional batch-wise learning, a pure self-paced way can further guarantee the efficiency and accuracy of SPML when dealing with small and biased samples. Furthermore, we construct a well-established SA-COVID dataset that contains 300 CT images with fine annotations. Extensive experiments on this dataset demonstrate that SPML consistently outperforms the state-of-the-art baselines. The SA-COVID dataset is publicly released at https://github.com/YishuLiu/SA-COVID.

Effects of ultrasonic treatment on the surface bacteria of Lyophyllum decastes during storage.

This study explores the relationship between the storage quality and bacterial microflora in the mushroom Lyophyllum decastes. The surface bacteria of L. decastes were separated by combining the traditional culture plate separation and 16S rRNA sequencing method, to study the effects of ultrasonic (US) treatment on the surface bacteria of L. decastes during storage. The results demonstrated that Pantoea agglomerans and Pseudomonas fluorescens were among the 15 culturable bacteria isolated with traditional plate method during storage, belonging to 2 phyla and 7 genera. US treatment could inhibit the growth and significantly increase cell membrane permeability, and contents extravasation in P. agglomerans, though its inhibitory effect on P. fluorescens was less. The 16S rRNA sequencing revealed, bacteria from 9 phyla and 35 genera were isolated, and P. fluorescens was the dominant species throughout the storage time. These results indicated that the composition of mushroom surface microflora of Control (CK) and US groups are similar, and the bacterial microflora networks analysis also showed a positive correlation. The KEGG annotation for the functional classification of the bacteria showed that a total of 328 pathways were acquired at the KEGG l3 level, and the relative abundance of membrane transport, amino acid metabolism, carbohydrate metabolism, and energy metabolism pathway was high. Moreover, the relative abundance of the surface bacteria of L. decastes also decreased. Hence, the US treatment had a better bacteriostatic effect, maintained the whiteness index and firmness, and improved the sensory quality of L. decastes during storage.

Flammutoxin, a Degradation Product of Transepithelial Electrical Resistance-Decreasing Protein, Induces Reactive Oxygen Species and Apoptosis in HepG2 Cells.

Proteins from Flammulina filiformis were prepared by sodium chloride extraction and fractionated by ammonium sulfate precipitation with increasing saturation degrees to obtain the protein fractions Ffsp-30, Ffsp-50, Ffsp-70, Ffsp-90, and Ffp-90. Among these protein fractions, Ffsp-50 possessed the most significant cytotoxic effect against three human gastrointestinal cancer cell lines, viz. HT-29, SGC-7901, and HepG2. SDS-PAGE and MALDI-TOF/TOF MS/MS analyses revealed that flammutoxin (FTX) was present as a dominating protein in Ffsp-50, which was further evidenced by HPLC-MS/MS determination. Furthermore, native FTX was purified from Ffsp-50 with a molecular weight of 26.78 kDa, exhibiting notable cytotoxicity against gastrointestinal cancer cell lines. Both Ffsp-50 and FTX exposure could enhance intercellular reactive oxygen species (ROS) generation and induce significant apoptosis in HepG2 cells. FTX was identified to be relatively conserved in basidiomycetes according to phylogenetic analysis, and its expression was highly upregulated in the primordium as well as the pileus of the fruiting body from the elongation and maturation stages, as compared with that in mycelium. Taken together, FTX could remarkably inhibit cell growth and induce ROS and apoptosis in HepG2 cells, potentially participating in the growth and development of the fruiting body. These findings from our investigation provided insight into the antigastrointestinal cancer activity of FTX, which could serve as a biological source of health-promoting and biomedical applications.

The Effect of Mitochondria on Ganoderma lucidum Growth and Bioactive Components Based on Transcriptomics.

Mitochondria are the power source of living cells and implicated in the oxidative metabolism. However, the effect of mitochondria on breeding is usually ignored in conventional research. In this study, the effect of mitochondria on Ganoderma lucidum morphology, yield, and main primary bioactive components was analyzed via structuring and comparing isonuclear alloplasmic strains. The crucial biological pathways were then explored based on the transcriptome. The results showed that isonuclear alloplasmic exhibited difference in mycelial growth rate in potato dextrose agar medium (PDA), basidiospore yield, and polysaccharide and triterpenoid content. Otherwise, mitochondria did not change colony and fruit body morphology, mushroom yield, or mycelial growth rate in solid-state fermentation cultivation material. The transcriptome data of two significant isonuclear alloplasmic strains S1 and S5 revealed that the involvement of differentially expressed genes (DEGs) was mainly in pentose and glucuronate interconversions, starch and sucrose metabolism, and steroid biosynthesis. The result was further confirmed by the other isonuclear alloplasmic strains. The above results further proved that mitochondria could affect the active components of G. lucidum. Our results provide information which will contribute to understanding of mitochondria and will be helpful for breeding improved varieties.

Palladium-catalysed construction of butafulvenes.

Butafulvene is a constitutional isomer of benzene, comprising a cyclobutene skeleton bearing two exocyclic conjugated methylene units. As a result of the intrinsic high strain energy and anti-aromaticity, the preparation of butafulvene compounds has been a fundamental issue for the development of butafulvene chemistry. Here an efficient palladium-catalysed coupling protocol involving propargylic compounds has been developed, providing a solid and versatile strategy for the rapid assembly of symmetric butafulvene derivatives. Based on mechanistic studies, two complementary mechanisms, both involving palladium catalysis, have been confirmed. With the mechanism unveiled, the synthesis of non-symmetric butafulvenes has also been achieved. Advantages of this strategy include tolerance to a wide range of propargylic molecules, mild reaction conditions, simple catalytic systems and easy scalability. The synthetic potential of the products as platform molecules for cyclobutene derivatives has also been demonstrated.

Ultrasonic treatment decreases Lyophyllum decastes fruiting body browning and affects energy metabolism.

Lyophyllum decastes is a common mushroom that is prone to browning during prolonged storage. In this study, the effects of ultrasonic treatment on metabolic gene expression, enzyme activity, and metabolic compounds related to L. decastes browning were investigated. Treatment of the fruiting body at 35 kHz and 300 W for 10 min reduced the browning index of L. decastes by 21.0 % and increased the L* value by 11.1 %. Ultrasonic treatment of the fruiting body resulted in higher levels of total phenols, flavonoids, and 9 kinds of amino acid with catalase (CAT) and peroxidase (POD) activities maintained at high levels. Higher cytochrome c oxidase (CCO), succinate dehydrogenase (SDH), phosphofructokinase (PFK), and pyruvate kinase (PK) activities may be ascribed to increased antioxidant capacity. Moreover, ultrasonication retained higher adenosine triphosphate (ATP) concentrations with an increased energy charge, while there were lower levels of adenosine diphosphate (ADP) and reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+), respectively. Meanwhile, lower lignin contents were observed, along with retarded polyphenol oxidase (PPO) and lipoxygenase (LOX) activities. Lower PPO activity reduced the fruiting body enzymatic browning rate through decreased expression of LdPpo1, LdPpo2, and LdPpo3 during storage at 4 °C for 16 days. This activity may be used to determine the effectiveness of ultrasonication.

Rhodium-Catalyzed Deuterated Tsuji-Wilkinson Decarbonylation of Aldehydes with Deuterium Oxide.

The recent surge in the applications of deuterated drug candidates has rendered an urgent need for diverse deuterium labeling techniques. Herein, an efficient Rh-catalyzed deuterated Tsuji-Wilkinson decarbonylation of naturally available aldehydes with D2O is developed. In this reaction, D2O not only acts as a deuterated reagent and solvent but also promotes Rh-catalyzed decarbonylation. In addition, decarbonylative strategies for the synthesis of terminal monodeuterated alkenes from α,ß-unsaturated aldehydes are within reach.

Effects of ambient air pollution, fresh fruit and vegetable intakes as well as maternal psychosocial stress on the outcome of newborn otoacoustic emission hearing screening.

BACKGROUND: Newborn hearing screening results indicated that more than 40% of the detected infants had no recognized risk factors. To determine whether maternal exposure to ambient air pollutants and experience of stressful life event, as well as lack of fresh fruit and vegetable during pregnancy are associated with the abnormal hearing development among newborns. METHODS: A total of 1193 newborns and their mothers were recruited in this study. Personal information and covariates were collected by face to face interview. Medical examination results of newborns and their mothers were extracted from medical record. We estimated personal air pollutant exposure level through inverse distance weighted method based on data from air quality monitoring stations and assessed the auditory development of newborns via distortion product otoacoustic emission (DPOAE). Unconditional logistic regression model was used to estimate the relationship between DPOAE screening result and the potential influential factors as well as the combined effect. RESULTS: The results indicated that PM10 exposure during the second trimester and stressful life event during the third trimester could increase the risk of not passing DPOAE test among newborns. However, frequent intakes of fruit and vegetable significantly reduced the risk. There was a synergetic interaction between PM10 exposure and stressful life event on neonatal hearing development. CONCLUSIONS: To alleviate abnormal auditory development among fetus, pregnant woman should decrease the exposures to ambient air pollutant and negative life event and at the same time, intake sufficient fresh fruit and vegetable.

Effects of Alcohol Extracts From Ganoderma resinaceum on Sleep in Mice Using Combined Transcriptome and Metabolome Analysis.

Ganoderma resinaceum is a valuable Chinese medicine. This study aimed to investigate whether a G. resinaceum alcohol extract (GRAE) improves sleep, and analyze the potential mechanism. After 30 days of continuous administration of GRAE at various doses, GRAE (1,000 mg/kg.bw) prolonged pentobarbital sodium-induced sleep, increased the rate of sleeping in mice treated with a subthreshold dose of pentobarbital sodium, and shortened sleep latency. The mice brain was analyzed using UPLC-MS/MS and RNA-sequencing. Metabolomics analysis revealed that 73 metabolites in the high-dose (HD) group had changed significantly, mainly in amino acids and their derivatives, especially the accumulation of L-glutamine and PGJ2 (11-oxo-15S-hydroxy-prosta-5Z, 9, 13E-trien-1-oic acid). Transcriptome analysis revealed 500 differential genes between HD and control groups, mainly enriched in neuroactive ligand-receptor interaction, amphetamine addiction, and cocaine addiction pathways. The conjoint analysis of the transcriptome and metabolome showed that the biosynthesis of L-glutamine might be regulated by Homer1, Homer3, and Grin3b. This suggests that GRAE may affect L-glutamine accumulation by regulating the expression of these genes. This study showed that GRAE may prolong the sleep time of mice by reducing the accumulation of L-glutamine and deepens our understanding of the regulatory network between certain genes and L-glutamine.

Anti-obesity effect of Auricularia heimuer fruiting body alcohol extraction on obese mice and crucial metabolite pathway analysis by liquid chromatography-tandem mass spectrometry.

In recent years, the increasing obese and overweight population has become a worldwide public health problem, as there is no effective medication to control obesity. Auricularia heimuer is rich in active substances that have potential biologically active functions. The anti-obesity effect and mechanism of Auricularia heimuer fruiting body alcohol extraction (AHA, 150-600 mg/kg·bw) was investigated in obese mice by assessing changes in endogenous liver metabolites using a liquid chromatography-tandem mass spectrometry approach. The aim of this study was to identify an effective food to control human obesity. AHA of 600 mg/kg·bw (HC) significantly decreased body weight and improved serum biochemistry indices. Sixty-eight liver metabolites were identified and significantly separated among the normal, high-fat diet (HFD), and HC groups. Moreover, the metabolic analysis revealed that HC significantly regulated specific metabolites in mice including amino acids, lipids, and carbohydrate compounds. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that HC was significantly involved in different metabolite pathways including arachidonic acid metabolism, galactose metabolism, carbohydrate digestion and absorption, linoleic acid metabolism, and starch and sucrose metabolism. Eight weeks after supplementing with HC, major metabolites in related pathways that were disrupted by an HFD were restored to normal levels, suggesting that HC had anti-obesity activity.

MBP-activated autoimmunity plays a role in arsenic-induced peripheral neuropathy and the potential protective effect of mecobalamin.

Intake excessive arsenic (As) is related to the occurrence of peripheral neuropathy. However, both the underlying mechanism and the preventive approach remain largely unknown. In the present study, As treatment significantly decreased the mechanical withdrawal threshold and increased the titer of anti-myelin basic protein antibody in rats, accompanied with damaged BNB. The levels of inflammatory cytokines and proteolytic enzymes were also significantly upregulated. However, administration of MeCbl in As-treated rats significantly reversed the decline in hindfoot mechanical withdrawal threshold, as well as BNB failure and sciatic nerve inflammation. Repeated As treatment in athymic nude mice indicated that sciatic nerve inflammation and mechanical hyperalgesia were T cell-dependent. These data implicated that MBP-activated autoimmunity and the related neuroinflammation probably contributed to As-induced mechanical hyperalgesia and MeCbl exerted a protective role probably via maintenance the integrity of BNB and inhibition of neuroinflammation.

The MADS-box transcription factor GlMADS1 regulates secondary metabolism in Ganoderma lucidum.

MADS-box transcription factors play crucial roles in regulating development processes and biosynthesis of secondary metabolites in eukaryotes. However, the role of MADS-box transcription factors vary among fungal species, and their function remains unclear in the medicinally and economically important fungus Ganoderma lucidum. In this study, we characterized a MADS-box gene, GlMADS1, in G. lucidum. Analyses using quantitative real-time polymerase chain reaction (qRT-PCR) showed that GlMADS1 expression levels were up-regulated from the mycelia to the primordia stage. In order to further evaluate the effect of MADS-box transcription factors on secondary metabolism, we utilized RNA interference (RNAi) to silence GlMADS1 in G. lucidum. Ganoderic acid (GA) and flavonoid contents were enhanced in GlMADS1-silenced strains, suggesting that GlMADS1 negatively regulates GA and flavonoid accumulation.

First Report of Cladosporium Blossom Blight Caused by Cladosporium cladosporioides on Calliandra haematocephala in China.

Calliandra haematocephala Hassk., commonly called red powder puff, is widely cultivated as an ornamental plant in Taiwan, Hainan, Guangdong and Fujian in China (CAS, 1988). The flowers are dark crimson with conspicuous stamens, which give them the appearance of powder-puffs. Blossom blight on C. haematocephala was first observed in early January 2019 on plants grown on the university campus as well as in parks in Fuzhou city, with nearly 80% of flowers on individual plants infected. At various locations in the city, disease incidence was 100%. Symptoms appeared as grayish green fungal growth on the stamens with the entire flower eventually turning black and covered with masses of fungal spores. Fifteen single spore isolates obtained from nine necrotic stamen samples were purified and cultured on Potato dextrose agar (PDA) plates at 24 ℃.The resultant fungal colonies were olivaceous-green to olivaceous-brown and had a velvet-like appearance. Conidiophores were smooth-walled, solitary, non-nodulose, and measuring 40 to 340 × 3 to 4 µm (n=50). Ramoconidia were cylindrical-oblong or slightly curved with 0 to 3 septa, and measuring 10 to 25 × 3 to 4 µm (n=50). Conidia were smooth-walled and prolifically produced in long chains. Terminal conidia were aseptate, subglobose, ovoid to limoniform, measuring 3 to 6 × 2 to 2.5 µm (n=50). Intercalary conidia were elliptical to limoniform or subcylindrical, aseptate, measuring 5 to 12 × 2.5 to 3 µm (n=50). On the basis of its morphology, the causal organism was identified as Cladosporium cladosporioides (Bensch et al. 2010). For molecular identification, pure cultures of five single-spore isolates were used for DNA extraction. A fragment in the ITS regions of the fungal rDNA, the ACT and the TEF1-α, was amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, and EF1728 F/EF1-986R. The DNA sequences obtained from all five isolates were identical. The resulting ITS (MK720012) and ACT (MN013164), and TEFl-α (MK752020) sequences from a representative isolate MRCIM19 were 98-100% identical to the C. cladosporioides accessions (ITS: MH863979, MG228421; ACT: HM148509, JF499878, HM148532; TEFl-α: JF499872). To test pathogenicity, a spore suspension (1×105 conidia/mL) was prepared from a seven- day- old culture of isolate MRCIM19 and 10 mL of the suspension was sprayed onto six flowers on each of three C. haematocephala plants. Sterile distilled water was sprayed onto three flowers of two plants as control. The inoculated flowers were covered with plastic bags which were removed two days post inoculation. Disease symptoms were recorded on each flower at 10 days post inoculation. Based on the morpho-molecular characters, the re-isolated fungus from the inoculated flowers was C. cladosporioides. This fungus was previously reported to cause blossom blight in strawberry in the USA and Korea (Gubler et al. 1999; Nam et al. 2015). Although it has been reported from many plants (Zhang 2003) in China, this is the first report of C. cladosporioides on C. haematocephala worldwide. References Bensch, K. et al. 2010. Stud Mycol. 67:1-94. Chinese Academy of Sciences (CAS), 1988. Flora Republicae Popularis Sinicae Editorial Committee, Beijing Sci. Press., 39: 38. Gubler, W. D. et al. 1999. Plant Dis. 83:400. Nam, M. H. et al. 2015. Microbiol. 43: 354-359. Zhang Z., Ed. 2003. Flora fungorum sinicorum, Vol. 14. Cladosporium, Fusicladium, Pyricularia. Beijing Science Press. 297.

Rhodium-catalyzed regio- and enantioselective allylic alkylation of pyrazol-5-ones with alkynes.

A rhodium-catalyzed asymmetric allylic alkylation of pyrazol-5-ones with internal alkynes is illustrated. In the presence of a chiral rhodium-hydride catalyst, functionalized heterocyclic products bearing an all-carbon quaternary stereogenic center were obtained in high yields with satisfactory enantioselectivities. This protocol also features good regiocontrol and a high atom economy without stoichiometric by-product formation.

Association of twenty-three plasma elements with fasting serum glucose among Chinese population from four areas with different pollution level.

BACKGROUND: Association between fasting serum glucose (FSG) and certain mineral elements has been extensively reported. Investigation regarding multi-element exposure among subjects with different exposure level is warranted to confirm the association and further explore dose-dependent relationship. METHODS: A total of 3488 participants were recruited from four counties of Hunan province, South China. Basic characteristics were collected by face to face interview and 23 elements in plasma were determined by inductively coupled plasma mass spectrometry. We applied fully adjusted generalized linear regression model and multivariable restricted cubic spline function to test the association and dose-response relationship of FSG with 23 elements. RESULTS: The results indicated that FSG was positively associated with plasma78selenium level [regression coefficient (ß), 0.001; 95 % confidence interval (CI), 0.001, 0.001] in a dose-dependent manner, robust to the adjustment for suspected covariates and stratification by age, gender, BMI and smoking status. A negative association was found between FSG and plasma 208lead (ß, -0.004; 95 % CI, -0.016, -0.002), 52chromium (ß, -0.002; 95 % CI, -0.004, -0.001) and 47titanium (ß, -0.001; 95 % CI, -0.002, -0.001). CONCLUSION: 78selenium was positively while 208lead, 52chromium and 47titanium were negatively associated with FSG in the present study. However, prospective studies are needed to confirm the results.

Cobalt-Catalyzed Regioselective Carboamidation of Alkynes with Imides Enabled by Cleavage of C-N and C-C Bonds.

Through the oxidative addition of cobalt into the N-C(O) bond of phthalimide and the subsequent decarbonylation, we describe an efficient cobalt-catalyzed intermolecular decarbonylative carboamidation of alkynes. High regioselectivities have been achieved for unsymmetrical alkynes (including aryl-alkyl or aryl-aryl) to deliver polysubstituted isoquinolones. To facilitate step economy, a three-component decarbonylative carboamidation of alkynes with phthalic anhydrides and amines has been demonstrated using the current cobalt catalysis.

Sex-specific influence of prenatal air pollutant exposure on neonatal neurobehavioral development and the sensitive window.

Increasing evidence indicates the adverse effect of air pollution exposure during pregnancy on neurologic development among children. However, the impact on neurobehavioral development in fetus remains unknown. In 2017, a total of 1193 mother-newborns pairs were enrolled in a birth cohort study in Changsha, China. Exposures to PM2.5, PM10, SO2, CO and NO2 were determined by using inverse distance weighted method based on local monitoring station data. Neurobehavioral measure was administered at 48-72 h postpartum by utilizing the neonatal behavioral neurological assessment (NBNA). Basic information and covariates were collected by face to face interview. Generalized linear regression and multivariable restricted cubic spline function were performed to explore the trimester-specific association and dose-response relationship of maternal air pollution exposure with NBNA score, respectively. In adjusted three-pollutant model, PM2.5 exposure in trimester 2 was negatively associated with behavior score (ß, -0.003; 95% CI, -0.006, -0.001) and the inverse relation was more pronounced in male infants. In addition, PM2.5 level in the 2nd trimester was negatively related to activetone score (ß, -0.012; 95% CI, -0.021, -0.002) in a dose-dependent manner for both genders. Collectively, our results demonstrated that prenatal exposure to PM2.5 was linked to poor neurobehavioral performance of newborns. The second trimester was the most sensitive time window for the developments of behavior and activetone, and male subject was more vulnerable as compared to females.