Immune to temperature interference sensor of carbon dioxide gas concentration based on a single modified fiber Bragg grating.

In this study, a novel method that can detect carbon dioxide (CO2) concentration and realize temperature immunity based on only one fiber Bragg grating (FBG) is proposed. The outstanding contribution lies in solving the temperature crosstalk issue of FBG and ensuring the accuracy of detection results under the condition of anti-temperature interference. To achieve immunity to temperature interference without changing the initial structure of FBG, the optical fiber cladding of FBG and adjacent optical fiber cladding at both ends of FBG are modified by a polymer coating. Moreover, a universal immune temperature demodulation algorithm is derived. The experimental results demonstrate that the temperature response sensitivity of the improved FBG is controlled within the range of 0.00407 nm/°C. Compared with the initial FBG (the temperature sensitivity of the initial FBG is 0.04 nm/°C), it decreases by nearly 10 times. Besides, the gas response sensitivity of FBG reaches 1.6 pm/ppm and has overwhelmingly ideal linearity. The detection error results manifest that the gas concentration error in 20 groups of data does not exceed 3.16 ppm. The final reproducibility research shows that the difference in detection sensitivity between the two sensors is 0.08 pm/ppm, and the relative error of linearity is 1.07%. In a word, the proposed method can accurately detect the concentration of CO2 gas and is efficiently immune to temperature interference. The sensor we proposed has the advantages of a simple production process, low cost, and satisfactory reproducibility. It also has the prospect of mass production.

[Research on the blended learning mode of "Microbial Breeding Experiments" based on WeChat].

With the rapid development of the internet technology, the blended learning mode plays a more and more important role in education reforms by integrating traditional classroom teaching and online learning. WeChat is the most popular Chinese social software, and its public platform is also suitable for mobile learning. Here, we report an application of the blended learning method based on WeChat public platforms in microbiology breeding experiment courses, using the site-directed mutagenesis of the green fluorescent protein (GFP) as an example. The learning process was divided into five modules: teaching design, learning resource preparation, pre-class learning, classroom learning, post-class review and evaluation. By introducing one mutation (Y66H) in mutagenic primers, the mutated GFP gene was amplified by PCR using pGFPuv as templates, followed by removal of the original plasmid template by Dpn1 digestion. Students can monitor the color changes from green to blue in the fluorescence emission of the mutated proteins. As a useful addition to classroom teaching, WeChat is suitable for students to use fragmented time to learn and improve teaching interaction. Learning assessment results revealed the blended learning environment improves students' study interests and self-learning abilities, thus achieving a fruitful teaching result.

Two bacterial group II phosphopantetheinyl transferases involved in both primary metabolism and secondary metabolism.

It is known that bacterial group II phosphopantetheinyl transferases (PPTases) usually phosphopantetheinylate acyl carrier proteins (ACPs) involved in the secondary metabolism. For example, a bacterial group II PPTase SchPPT has been known to phosphopantetheinylate only ACPs involved in secondary metabolism, such as scn ACP0-2 and scn ACP7. In this study, we found two bacterial group II PPTases, Hppt and Sppt, could phosphopantetheinylate not only scn ACP0-2 and scn ACP7, but also sch FAS ACP, an ACP involved in primary metabolism. Swapping of the N terminus and C terminus of PPTases showed that (i) both the hybrids Hppt-Sppt and Sppt-Hppt could phosphopantetheinylate sch FAS ACP but not scn ACP0-2; (ii) both the hybrids Sppt-SchPPT and SchPPT-Sppt lost abilities to phosphopantetheinylate sch FAS ACP and scn ACP0-2. Hppt and Sppt represent group II PPTases which phosphopantetheinylate both ACPs involved in primary metabolism and ACPs involved in secondary metabolism.

[Study on an inquiry-based teaching case in genomics curriculum: identifying virulence factors of Escherichia coli by using comparative genomics].

Genomics is the core subject of various "omics" and it also becomes a topic of increasing interest in undergraduate curricula of biological sciences. However, the study on teaching methodology of genomics courses was very limited so far. Here we report an application of inquiry-based teaching in genomics courses by using virulence factors of Escherichia coli as an example of comparative genomics study. Specially, students first built a multiple-genome alignment of different E. coli strains to investigate the gene conservation using the Mauve tool; then putative virulence factor genes were identified by using BLAST tool to obtain gene annotations. The teaching process was divided into five modules: situation, resources, task, process and evaluation. Learning-assessment results revealed that students had acquired the knowledge and skills of genomics, and their learning interest and ability of self-study were also motivated. Moreover, the special teaching case can be applied to other related courses, such as microbiology, bioinformatics, molecular biology and food safety detection technology.

[Comparative genomics and evolutionary analysis of CRISPR loci in acetic acid bacteria].

The clustered regularly interspaced short palindromic repeat (CRISPR) is a widespread adaptive immunity system that exists in most archaea and many bacteria against foreign DNA, such as phages, viruses and plasmids. In general, CRISPR system consists of direct repeat, leader, spacer and CRISPR-associated sequences. Acetic acid bacteria (AAB) play an important role in industrial fermentation of vinegar and bioelectrochemistry. To investigate the polymorphism and evolution pattern of CRISPR loci in acetic acid bacteria, bioinformatic analyses were performed on 48 species from three main genera (Acetobacter, Gluconacetobacter and Gluconobacter) with whole genome sequences available from the NCBI database. The results showed that the CRISPR system existed in 32 species of the 48 strains studied. Most of the CRISPR-Cas system in AAB belonged to type I CRISPR-Cas system (subtype E and C), but type II CRISPR-Cas system which contain cas9 gene was only found in the genus Acetobacter and Gluconacetobacter. The repeat sequences of some CRISPR were highly conserved among species from different genera, and the leader sequences of some CRISPR possessed conservative motif, which was associated with regulated promoters. Moreover, phylogenetic analysis of cas1 demonstrated that they were suitable for classification of species. The conservation of cas1 genes was associated with that of repeat sequences among different strains, suggesting they were subjected to similar functional constraints. Moreover, the number of spacer was positively correlated with the number of prophages and insertion sequences, indicating the acetic acid bacteria were continually invaded by new foreign DNA. The comparative analysis of CRISR loci in acetic acid bacteria provided the basis for investigating the molecular mechanism of different acetic acid tolerance and genome stability in acetic acid bacteria.

Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin.

Avilamycin is one of EU-approved antimicrobial agents in feed industry to inhibit the growth of multidrug-resistant Gram-positive bacteria. Here, we applied a process of combining ribosome engineering and genome shuffling to achieve rapid improvement of avilamycin production in Streptomyces viridochromogenes AS 4.126. The starting mutant population was generated by (60)Co γ-irradiation treatments of the spores. After five rounds of protoplast fusion with streptomycin-resistance screening, an improved recombinant E-219 was obtained and its yield of avilamycin reached 1.4 g/L, which was increased by 4.85-fold and 36.8-fold in comparison with that of the shuffling starter Co γ-316 and the ancestor AS 4.126. Furthermore, the mechanism for the improvement of shuffled strains was investigated. Recombinants with enhanced streptomycin resistance exhibited significantly higher avilamycin production and product resistance, probably due to the mutations in the ribosome protein S12. The morphological difference between the parent mutant and shuffled recombinant was observed in conidiospore, and hyphae pellets. The presence of genetic diversity among shuffled populations with varied avilamycin productivity was confirmed by randomly amplified polymorphic DNA analysis. In summary, our results demonstrated that genome shuffling combined with ribosome engineering was a powerful approach for molecular breeding of high-yield industrial strains.

Genome sequencing and genetic breeding of a bioethanol Saccharomyces cerevisiae strain YJS329.

BACKGROUND: Environmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits. RESULTS: We explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified. CONCLUSIONS: Through comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.

[Establishment of indices for soybean low temperature damages in key growth stage in high cold region]. / é«˜å¯’åŒºå¤§è±†å ³é”®ç”Ÿè‚²æœŸä½Žæ¸©å†·å®³æŒ‡æ ‡æž„å»º.

Establishment of indices for low temperature damage of soybeans is important for systematically analyzing the adaptation strategies to climate change and collaborative adaptation technology for disaster prevention and mitigation and other stresses in high cold region. Based on historical data of low temperature damage and phenophase of soybean from 1980 to 2020 and daily temperature data from 78 meteorological stations in Heilongjiang Province, we used GIS to match the phenophase and meteorological data, by considering the accumulated temperature anomaly in different growth stages, and constructed a comprehensive soybean low temperature damages index (CSCDI) in high cold region. Using K-S distribution fitting test and the lower limit value of confidence intervals, we constructed the level index of soybean low temperature damage. The results showed that the CSCDI lower limits of mild, moderate and severe low temperature damage were 0.061, 0.115 and 0.237 from sowing to emergence stage, were 0.072, 0.152 and 0.312 from emergence to flowering, and 0.133, 0.245 and 0.412 from flowering to maturity, respectively. The time distribution of soybean low temperature damage inversed by CSCDI in Heilongjiang Province was consistent with the historical disaster records. The spatial distribution showed obvious latitude characteristics, with the frequency of low temperature damage increasing gradually from south to north.

Comparative transcriptomic analyzes of human lung epithelial cells infected with wild-type SARS-CoV-2 and its variant with a 12-bp missing in the E gene.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused a global outbreak of coronavirus disease 2019 (COVID-19) pandemic. To elucidate the mechanism of SARS-CoV-2 replication and immunogenicity, we performed a comparative transcriptome profile of mRNA and long non-coding RNAs (lncRNAs) in human lung epithelial cells infected with the SARS-CoV-2 wild-type strain (8X) and the variant with a 12-bp deletion in the E gene (F8). In total, 3,966 differentially expressed genes (DEGs) and 110 differentially expressed lncRNA (DE-lncRNA) candidates were identified. Of these, 94 DEGs and 32 DE-lncRNAs were found between samples infected with F8 and 8X. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzes revealed that pathways such as the TNF signaling pathway and viral protein interaction with cytokine and cytokine receptor were involved. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to the immune response, which might explain the different replication and immunogenicity properties of the 8X and F8 strains. These results provide a useful resource for studying the pathogenesis of SARS-CoV-2 variants.

The complete chloroplast genome sequences of Hippuris vulgaris (Plantaginaceae).

Two complete chloroplast genomes of Hippuris vulgaris (H. vulgaris_A and H. vulgaris_B), representing two distinct clades in China, were sequenced and assembled in this study. The circular genomes were 152,763 and 152,713 bp in length and exhibit a typical quadripartite structure of the large single-copy (LSC, 82,983/82,949 bp) and small single-copy (SSC, 18,294/18,278 bp) regions, separated by a pair of inverted repeats (IRs, both 25,743 bp). Both two cp genomes identically contain 133 genes, including 88 protein-coding genes, 37 tRNA, and eight rRNA genes. The phylogenetic analysis within Plantaginaceae demonstrated Hippuris an independent clade included in the expanded Plantaginaceae.

Characterization of the complete chloroplast genome of Sparganium glomeratum (Typhaceae) from Jilin Province, China and phylogenetic analysis.

The complete chloroplast genome of Sparganium glomeratum was sequenced and assembled in this study. The circular genome is 160,391 bp in length and exhibits a typical quadripartite structure with a large single-copy (LSC, 87,660 bp) and small single-copy (SSC, 18,721 bp) regions, separated by a pair of inverted repeats (IRs, 27,005 bp). The cp genome contains 113 unique genes, including 79 protein-coding, 30 tRNA, and four rRNA genes. The phylogenetic analysis within the Poales showed that Sparganium is monophyletic and most closely related to Typha. Within Sparganium, S. glomeratum is sister to the clade of S. stoloniferum and S. euricarpum. The work reported here will provide useful information for the evolutionary studies on the genus of Sparganium.

Effect of thromboprophylaxis on the incidence of venous thromboembolism in surgical patients with colorectal cancer: a meta-analysis.

INTRODUCTION: This study evaluates the role of thromboprophylaxis in venous thromboembolism (VTE) incidence in colorectal cancer (CRC) surgical patients. EVIDENCE ACQUISITION: Literature search was conducted in electronic databases and studies (randomized controlled trials, or prospective/retrospective cohorts) were selected if they reported the incidence of VTE in CRC patients who underwent any type of open or laparoscopic surgery. Random-effects meta-analyses were performed to obtain pooled incidence estimates; or odds ratios (OR) of incidence between prophylaxis and no-prophylaxis groups; or between VTE and non-VTE patients to identify risk factors. EVIDENCE SYNTHESIS: Twenty-four studies (804,003 patients) were included. Prophylaxis was performed mainly with low molecular weight heparins. Odds of VTE were significantly lower in prophylactic than in non-prophylactic patients (OR 0.42 [95% CI: 0.28, 0.63]; P<0.00001). Incidence of radiological and symptomatic VTE in patients with prophylaxis was 9.7% [95% CI: 8.6, 10.8] and 1.3% [95% CI: 0.7, 2.0] respectively. Odds of bleeding were higher in patients with prophylaxis (OR: 3.37 [95% CI: 1.05, 10.8]; P=0.04). Incidence of bleeding in patients with and without prophylaxis was 4.3% [95% CI: 3.2, 5.4] and 1.2% [95% CI: 0.02, 2.4] respectively. Operative time, anesthesia duration, and hospital stay were longer in patients with VTE. Obesity, disseminated cancer, chemotherapy, steroid use, emergency case status, advanced stage cancer, hypoalbuminemia, postoperative infection/sepsis, and history of VTE are identified as important risk factors for VTE incidence. CONCLUSIONS: Thromboprophylaxis reduces the incidence of VTE in CRC surgical patients but may increase the chances of bleeding. Several risk factors can influence VTE incidence.

Expression of PD-1/PD-L1 in head and neck squamous cell carcinoma and its clinical significance.

OBJECTIVE: To investigate the role of programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and P16 in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: A total of 95 paraffin-embedded samples of tumorous tissue of HNSCC were collected. Expression levels of PD-1, PD-L1, and P16 were determined by immunohistochemistry. RESULTS: A significantly higher proportion of PD-1 among patients infected with the human papillomavirus was found. PD-L1 expression is closely associated with the primary site of the tumor, postoperative recurrence, survival, PD-1 expression and P16 expression. Univariable analysis indicated that T stage, N stage, tumor node metastasis stage, tumor differentiation, and PD-L1 expression were all shown to be prognostic variables for overall survival in patients with HNSCC. In the multivariate analysis, only N stage (P = 0.010) and PD-L1 expression (P = 0.001) were found to be independent prognostic variables for overall survival. In addition, for disease recurrence, multivariate analysis showed that only PD-L1 expression was the associated independent risk factor. For the patients with negative PD-L1 expression, Kaplan-Meier analysis revealed that they had significantly worse outcomes in terms of overall survival (P = 0.001). Similarly, compared with the patients with positive PD-L1 expression, those with negative PD-L1 expression had a higher probability of recurrence (P = 0.026). CONCLUSIONS: The expression of PD-L1, PD-1, and P16 in HNSCC is significantly correlated. Human papillomavirus infection (P16 positive) is negatively related to postoperative recurrence. HNSCC patients with positive PD-L1/PD-1 expression tend to have better overall survival outcomes and lower probability of recurrence, providing more evidence for the PD-l-targeted immunotherapy of HNSCC.

Functional characterization of Streptomyces coelicolor FtsY.

This study indicated that the N-terminus was dispensable for FtsY GTPase activity, and that the N-domain plays an essential role in the GTPase activity of the NG domain. In addition, the S.scoelicolor FtsY was able to restore function in an E. coli mutant. However, its NG domain was unable to play any roles.

Factors affecting genotyping success in giant panda fecal samples.

Fecal samples play an important role in giant panda conservation studies. Optimal preservation conditions and choice of microsatellites for giant panda fecal samples have not been established. In this study, we evaluated the effect of four factors (namely, storage type (ethanol (EtOH), EtOH -20 °C, 2-step storage medium, DMSO/EDTA/Tris/salt buffer (DETs) and frozen at -20 °C), storage time (one, three and six months), fragment length, and repeat motif of microsatellite loci) on the success rate of microsatellite amplification, allelic dropout (ADO) and false allele (FA) rates from giant panda fecal samples. Amplification success and ADO rates differed between the storage types. Freezing was inferior to the other four storage methods based on the lowest average amplification success and the highest ADO rates (P < 0.05). The highest microsatellite amplification success was obtained from either EtOH or the 2-step storage medium at three storage time points. Storage time had a negative effect on the average amplification of microsatellites and samples stored in EtOH and the 2-step storage medium were more stable than the other three storage types. We only detected the effect of repeat motif on ADO and FA rates. The lower ADO and FA rates were obtained from tri- and tetra-nucleotide loci. We suggest that freezing should not be used for giant panda fecal preservation in microsatellite studies, and EtOH and the 2-step storage medium should be chosen on priority for long-term storage. We recommend candidate microsatellite loci with longer repeat motif to ensure greater genotyping success for giant panda fecal studies.

Hispidin induces autophagic and necrotic death in SGC-7901 gastric cancer cells through lysosomal membrane permeabilization by inhibiting tubulin polymerization.

Hispidin and its derivatives are widely distributed in edible mushrooms. Hispidin is more cytotoxic to A549, SCL-1, Bel7402 and Capan-1 cancer cells than to MRC5 normal cells; by contrast, hispidin protects H9c2 cardiomyoblast cells from hydrogen peroxide-induced or doxorubicin-induced apoptosis. Consequently, further research on how hispidin affects normal and cancer cells may help treat cancer and reduce chemotherapy-induced side effects. This study showed that hispidin caused caspase-independent death in SGC-7901 cancer cells but not in GES-1 normal cells. Hispidin-induced increases in LC3-II occurred in SGC-7901 cells in a time independent manner. Cell death can be partially inhibited by treatment with ATG5 siRNA but not by autophagy or necroptosis inhibitors. Ultrastructural evidence indicated that hispidin-induced necrotic cell death involved autophagy. Hispidin-induced lysosomal membrane permeabilization (LMP) related to complex cell death occurred more drastically in SGC-7901 cells than in GES-1 cells. Ca2+ rather than cathepsins from LMP contributed more to cell death. Hispidin induced microtubule depolymerization, which can cause LMP, more drastically in SGC-7901 cells than in GES-1 cells. At 4.1 µM, hispidin promoted cell-free tubulin polymerization but at concentrations higher than 41 µM, hispidin inhibited polymerization. Hispidin did not bind to tubulin. Alterations in microtubule regulatory proteins, such as stathmin phosphorylation at Ser16, contributed to hispidin-induced SGC-7901 cell death. In conclusion, hispidin at concentrations higher than 41 µM may inhibit tubulin polymerization by modulating microtubule regulatory proteins, such as stathmin, causing LMP and complex SGC-7901 cell death. This mechanism suggests a promising novel treatment for human cancer.

Whole genome analysis of non-optimal codon usage in secretory signal sequences of Streptomyces coelicolor.

Non-optimal (rare) codons have been suggested to reduce translation rate and facilitate secretion in Escherichia coli. In this study, the complete genome analysis of non-optimal codon usage in secretory signal sequences and non-secretory sequences of Streptomyces coelicolor was performed. The result showed that there was a higher proportion of non-optimal codons in secretory signal sequences than in non-secretory sequences. The increased tendency was more obvious when tested with the experimental data of secretory proteins from proteomics analysis. Some non-optimal codons for Arg (AGA, CGU and CGA), Ile (AUA) and Lys (AAA) were significantly over presented in the secretary signal sequences. It may reveal that a balanced non-optimal codon usage was necessary for protein secretion and expression in Streptomyces.

Genome Sequencing and Evolutionary Analysis of Marine Gut Fungus Aspergillus sp. Z5 from Ligia oceanica.

Aspergillus sp. Z5, isolated from the gut of marine isopods, produces prolific secondary metabolites with new structure and bioactivity. Here, we report the draft sequence of the approximately 33.8-Mbp genome of this strain. To the best of our knowledge, this is the first genome sequence of Aspergillus strain isolated from marine isopod Ligia oceanica. The phylogenetic analysis supported that this strain was closely related to A. versicolor, and genomic analysis revealed that Aspergillus sp. Z5 shared a high degree of colinearity with the genome of A. sydowii. Our results may facilitate studies on discovering the biosynthetic pathways of secondary metabolites and elucidating their evolution in this species.

Nanosphere-in-a-nanoegg: damping the high-order modes induced by symmetry breaking.

We study the optical properties of the nanosphere-in-a-nanoegg structure (NSNE) by the three-dimensional finite difference time domain method. We demonstrate the suppression of the high-order plasmon modes in NSNE, which is induced by the plasmon interaction between the inner nanosphere and the outer nanoegg shell. A two-layer plasmon hybridization model is presented to explain this mechanism. The results we showed for plasmon mode suppression would be important to the design of the metal plasmonic devices. In addition, due to high tunable plasmon resonances in the near-infrared region (700 to 1,300 nm) with sub-100-nm size, NSNE can serve as a good substitute for the Au-silica-Au multilayer nanoshells in biological applications. Furthermore, compared with the Au-silica-Au nanoshells, NSNE has the advantage that the strong field enhancement can be achieved at the outer surface of the Au shell.

The Effect of the Modified Eighth Section of Eight-Section Brocade on Osteoporosis in Postmenopausal Women: A Prospective Randomized Trial.

Osteoporosis and related fragility fractures represent a serious and global public health problem. To evaluate whether the modified eighth section of Eight-section Brocade (MESE) exercise could improve the symptom and indexes associated with osteoporosis in postmenopausal women. Guangzhou and Liuzhou hospital of traditional Chinese medicine in China. Women (n = 198) aged 50 to 75 years were randomized into Control, Ca, MESE, and MESE + Ca. Subjects in Ca and MESE groups were separately asked to consume thrice daily Calcium Carbonate Chewable D3 tablet and to perform thrice daily MESE exercise by 7 repetitions per time for 12 months. Subjects in MESE + Ca group performed such the combined treatment project for 12 months. Body height and Hospital for Special Surgery (HSS) scores of both knees, chronic back pain visual analogue scale scores (VAS), bone mineral density (BMD) at L2 to L4 and the left femoral neck, 3-feet Up and Go Test (3') and one-leg Stance (OLS). In our study, the improvement in chronic back pain of the patients in Ca, MESE, and MESE + Ca group was better than that in control group. There was 1.9% and 1.7%, 2.3%, and 2.1% net profit in left femoral neck and lumbar BMD after the treatment for 12 months in MESE and MESE + Ca groups. For the balance capacity, the subjects in MESE and MESE + Ca groups secured much better performance than those in Ca and control group after the treatment for 12 months (P < 0.001, P < 0.001). The treatment of MESE exercise is the most effective for the improvement of the symptom and indexes in postmenopausal women. Importantly, the low attrition and the high exercise compliance indicate that MESE exercise is safe, feasible, and well tolerated by postmenopausal women.