Hysteresis effects of different levels of storm flooding on susceptible enteric infectious diseases in a central city of China.

BACKGROUND: Recently, attention has focused on the impact of global climate change on infectious diseases. Storm flooding is an extreme weather phenomenon that not only impacts the health of the environment but also worsens the spread of pathogens. This poses a significant challenge to public health security. However, there is still a lack of research on how different levels of storm flooding affect susceptible enteric infectious diseases over time. METHODS: Data on enteric infectious diseases, storm flooding events, and meteorology were collected for Changsha, Hunan Province, between 2016 and 2020. The Wilcoxon Rank Sum Test was used to identify the enteric infectious diseases that are susceptible to storm flooding. Then, the lagged effects of different levels of storm flooding on susceptible enteric infectious diseases were analyzed using a distributed lag nonlinear model. RESULTS: There were eleven storm flooding events in Changsha from 2016 to 2020, concentrated in June and July. 37,882 cases of enteric infectious diseases were reported. During non-flooding days, the daily incidence rates of typhoid/paratyphoid and bacillary dysentery were 0.3/100,000 and 0.1/100,000, respectively. During flooding days, the corresponding rates increased to 2.0/100,000 and 0.8/100,000, respectively. The incidence rates of both diseases showed statistically significant differences between non-flooding and flooding days. Correlation analysis shows that the best lags for typhoid/paratyphoid and bacillary dysentery relative to storm flooding events may be 1 and 3 days. The results of the distributed lag nonlinear model showed that typhoid/paratyphoid had the highest cumulative RR values of 2.86 (95% CI: 1.71-4.76) and 8.16 (95% CI: 2.93-22.67) after 4 days of general flooding and heavy flooding, respectively; and bacillary dysentery had the highest cumulative RR values of 1.82 (95% CI: 1.40-2.35) and 3.31 (95% CI: 1.97-5.55) after 5 days of general flooding and heavy flooding, respectively. CONCLUSIONS: Typhoid/paratyphoid and bacillary dysentery are sensitive enteric infectious diseases related to storm flooding in Changsha. There is a lagging effect of storm flooding on the onset of typhoid/paratyphoid and bacillary dysentery, with the best lagging periods being days 1 and 3, respectively. The cumulative risk of typhoid/paratyphoid and bacillary dysentery was highest at 4/5 days lag, respectively. The higher of storm flooding, the higher the risk of disease, which suggests that the authorities should take appropriate preventive and control measures before and after storm flooding.

Gestational diabetes mellitus suppresses fetal testis development in mice†.

The prevalence of gestational diabetes mellitus (GDM) is increasing rapidly. In addition to the metabolic disease risks, GDM might increase the risks of cryptorchidism in children. However, its mechanism involved in abnormalities of the male reproductive system is still unclear. The purpose of this study was to study the effects of GDM on the development of mouse fetal Leydig cells (FLCs) and Sertoli cells (SCs). Pregnant mice were treated on gestational days 6.5 and 12.5 with streptozotocin (100 mg/kg) or vehicle (sodium citrate buffer). Leydig cell and SC development and functions were evaluated by investigating serum testosterone levels, cell number and distribution, genes, and protein expression. GDM decreased serum testosterone levels, the anogenital distance, and the level of desert hedgehog in SCs of testes of male offspring. FLC number was also decreased in testes of GDM offspring by delaying the commitment of stem Leydig cells into the Leydig cell lineage. RNA-seq showed that FOXL2, RSPO1/ß-catenin signaling was activated and Gsk3ß signaling was inhibited in GDM offspring testis. In conclusion, GDM disrupted reproductive tract and testis development in mouse male offspring via altering genes related to development.

Quantitative analysis of intraoperative electrocorticography mirrors histopathology and seizure outcome after epileptic surgery in children.

BACKGROUND: Epileptic surgery is the potentially curative treatment for children with refractory seizures. The study aimed to quantify and analyze high frequency oscillation (HFO) ripples and interictal epileptiform discharges (EDs) in intraoperative electrocorticography (ECoG) between malformation of cortical dysplasia (MCD) and non-MCD children with MRI-lesional focal epilepsy, and evaluate of seizure outcomes after epileptic surgery. METHODS: The intraoperative ECoG was performed before and after lesionectomy. Quantifications of HFO ripples and interictal EDs of ECoG by frequency, amplitude, and foci of intraoperative ECoG were performed based on electrode location, and the characteristics of ECoG recordings were analyzed in each patient based on their histopathology. Seizure outcome after surgery according to their quantitative ECoG findings was analyzed. RESULTS: Frequency of EDs and HFO ripple rates in preresection ECoG were significantly higher in children with MCD compared with non-MCD (p = 0.018 and p = 0.002, respectively). Higher frequencies of EDs and ripple rates in preresection ECoG were observed in residual seizures than in seizure-free children (p = 0.045 and p = 0.005, respectively). Clinically, children with residual seizures after surgery were significantly younger at the onset, had a trend of higher seizure frequency and higher spike frequency of presurgical videoEEG. CONCLUSION: Our results suggested that quantification of intraoperative ECoG predicted seizure outcomes and reflected different ED pattern and frequencies between MCD and non-dysplastic histopathology among children who underwent resective epileptic surgery. The results of our study were encouraging and indicated that intraoperative ECoG improved the outcomes of surgery in children with epilepsy.

[Analysis of genetic variants in four children with congenital hyperinsulinemia].

OBJECTIVE: To explore the genetic basis of four children with congenital hyperinsulinemia (CHI). METHODS: The four children were subjected to high-throughput whole exome sequencing (WES). Candidate variants were validated by Sanger sequencing. RESULTS: WES analysis has identified 4 variants in the ABCC8 gene and 1 variant in GLUD1, including a ABCC8 c.382G>A variant in case 1, compound heterozygous c.698T>C and c.4213G>A variants of the ABCC8 gene concomitant with a de novo 14.9 Mb microduplication of chromosome 15 in case 2, and ABCC8 c.331G>A variant in case 3, and de novo c.955T>C variant of the GLUD1 gene in case 4. Of these, c.698T>C of the ABCC8 gene and c.955T>C of the GLUD1 gene were unreported previously. Based on the American College of Medical Genetics and Genomics guidelines, the c.382G>A(p.Glu128Lys), c.698T>C(p.Met233Thr) and c.4213G>A(p.Asp1405Asn) variants of ABCC8 gene and c.955T>C(p.Tyr319His) variant of GLUD1 gene were predicted to be likely pathogenic(PM1+PM2+PP3+PP4, PM1+PM2+PM5+PP3+PP4, PM1+PM2+PP3+PP4 and PS1+PM1+PM2+PP3), and the c.331G>A (p.Gly111Arg) variant of ABCC8 gene was predicted to be uncertain significance(PM1+PM2+PP4). CONCLUSION: The variants of the ABCC8 and GLUD1 genes probably underlay the pathogenesis of CHI in the four patients. Above results have facilitated clinical diagnosis and genetic counseling for the affected families.

A novel pathogenic frameshift variant unmasked by a large de novo deletion at 13q21.33-q31.1 in a Chinese patient with neuronal ceroid lipofuscinosis type 5.

BACKGROUND: Neuronal ceroid lipofuscinosis type 5 (CLN5) is a rare form of neuronal ceroid lipofuscinoses (NCLs) which are a group of inherited neurodegenerative diseases characterized by progressive intellectual and motor deterioration, visual failure, seizures, behavioral changes and premature death. CLN5 was initially named Finnish variant late infantile NCL, it is now known to be present in other ethnic populations and with variable age of onset. Few CLN5 patients had been reported in Chinese population. CASE PRESENTATION: In this paper, we report the symptoms of a Chinese patient who suffer from developmental regression and grand mal epilepsy for several years. The DNA was extracted from peripheral blood of proband and both parents, and then whole exome sequencing was performed using genomic DNA. Both sequence variants and copy number variants (CNVs) were analyzed and classified according to guidelines. As the result, a novel frameshift mutation c.718_719delAT/p.Met240fs in CLN5 and a de novo large deletion at 13q21.33-q31.1 which unmasked the frameshift mutation were identified in the proband. Despite the large de novo deletion, which can be classified as a pathogenic copy number variant (CNV), the patient's clinical presentation is mostly consistent with that of CLN5, except for early developmental delay which is believed due to the large deletion. Both variants were detected simultaneously by exome sequencing. CONCLUSIONS: This is the first report of whole gene deletion in combination with a novel pathogenic sequence variant in a CLN5 patient. The two mutations detected with whole exome sequencing simultaneously proved the advantage of the sequencing technology for genetic diagnostics.

A novel variant of IHH in a Chinese family with brachydactyly type 1.

BACKGROUND: Brachydactyly type A1(BDA-1) is an autosomal dominant disorder which is caused by heterozygous pathogenic variants in a specific region of the N-terminal active fragment of Indian Hedgehog (IHH). The disorder is mainly characterized by shortening or missing of the middle phalanges. In this study, Our purpose is to identify the pathogenic variations associated with BDA-1 involved in a five-generation Chinese family. METHODS: A BDA-1 family with 8 affected and 14 unaffected family members was recruited. Whole exome sequencing (WES) was performed to identify the pathogenic variant in the proband, and which was later confirmed and segregated by Sanger sequencing. The significance of variants were assessed using several molecular and bioinformatics analysis methods. RESULTS: We uncovered a novel heterozygous missense variant c.299A > G (p.D100G) at the mutational hotspot of IHH gene following whole-exome sequencing of a Chinese family with BDA-1. The variant co-segregated with BDA-1 in the pedigree, showed 100% penetrance for phalange phenotype with variable expressivity. CONCLUSIONS: In conclusion, this study reports a five-generation Chinese family with BDA-1 due to a novel pathogenic variant (c.299A > G (p.D100G)) of IHH and expands the clinical and genetic spectrum of BDA-1.

Intrauterine exposure to hyperglycemia retards the development of brown adipose tissue.

Brown adipose tissue (BAT) is an exclusive tissue of nonshivering thermogenesis. It is fueled by lipids and glucose and involved in energy and metabolic homeostasis. Intrauterine exposure to hyperglycemia during gestational diabetes mellitus may result in abnormal fetal development and metabolic phenotypes in adulthood. However, whether intrauterine hyperglycemia influences the development of BAT is unknown. In this study, mouse embryos were exposed to the intrauterine hyperglycemia environment by injecting streptozocin into pregnant mice at 1 d post coitum (dpc). The structure of BAT was examined by hematoxylin and eosin staining and immunohistochemical analysis. The glucose uptake in BAT was measured in vivo by [18F]-fluoro-2-deoxyglucose-micro-positron emission tomography. The gene expression in BAT was determined by real-time PCR, and the 5'-C-phosphate-G-3' site-specific methylation was quantitatively analyzed. Intrauterine hyperglycemia exposure resulted in the impaired structure of BAT and decreased glucose uptake function in BAT in adulthood. The expressions of the genes involved in thermogenesis and mitochondrial respiratory chain in BAT, such as Ucp1, Cox5b, and Elovl3, were down-regulated by intrauterine hyperglycemia exposure at 18.5 dpc and at 16 wk of age. Furthermore, higher methylation levels of Ucp1, Cox5b, and Elovl3 were found in offspring of mothers with streptozotocin-induced diabetes. Our results provide the evidence for enduring inhibitory effects of intrauterine hyperglycemia on BAT development in offspring. Intrauterine hyperglycemia is associated with increased DNA methylation of the BAT specific genes in offspring, which support an epigenetic involvement.-Yu, D.-Q., Lv, P.-P., Yan, Y.-S., Xu, G.-X., Sadhukhan, A., Dong, S., Shen, Y., Ren, J., Zhang, X.-Y., Feng, C., Huang, Y.-T., Tian, S., Zhou, Y., Cai, Y.-T., Ming, Z.-H., Ding, G.-L., Zhu, H., Sheng, J.-Z., Jin, M., Huang, H.-F. Intrauterine exposure to hyperglycemia retards the development of brown adipose tissue.

Microarray-based identification of genes associated with prognosis and drug resistance in ovarian cancer.

The outcome for patients with ovarian cancer (OC) is poor because of drug resistance. Therefore, identification of factors that affect drug resistance and prognosis in OC is needed. In the present study, we identified 131 genes significantly dysregulated in 90 platinum-resistant OC tissues compared with 197 sensitive tissues, of which 30 were significantly associated with disease-free survival (DFS; n = 16), overall survival (OS; n = 6), or both (n = 8) in 489 OC patients of the The Cancer Genome Atlas cohort. Of these 30 genes, 17 were significantly upregulated and 13 were downregulated in the 90 resistant tissues, and with one exception, all of the up-/downregulated genes in resistant tissues were predictors of shorter DFS or/and OS. LAX1, MECOM, and PDIA4 were independent risk factors for DFS, and KLF1, SLC7A11, and PDIA4 for OS; combining these genes provided more accurate predictions for DFS and OS than any of the genes used individually. We further verified downregulation of PDIA4 protein in 51 specimens of patients with OC (24 drug resistant's and 27 sensitive's), which confirmed that downregulated PDIA4 predicted DFS and OS. PDIA4 also consistently predicted OS in a larger sample of 1656 patients with OC. These 30 genes, particularly the PDIA4, could be therapeutic targets or biomarkers for managing OC.

The prevalence and molecular characterization of (뫧)0 -thalassemia and hereditary persistence of fetal hemoglobin in the Chinese Zhuang population.

OBJECTIVE: To reveal the prevalence and molecular characterization of (δß)0 -thalassemia [(δß)0 -thal] and hereditary persistence of fetal hemoglobin (HPFH) in the Chinese Zhuang population. METHODS: A total of 105 subjects with fetal hemoglobin (Hb F) level ≥5% from 14 204 unrelated ones were selected for the study. Multiplex ligation dependent probe amplification was firstly used to analyze dosage changes of the ß-globin gene cluster for associated with (δß)0 -thal and HPFH mutations. The gap polymerase chain reaction was then performed to identify the deletions using the respective flanking primers. Hematologic data were recorded and correlated with the molecular findings. RESULTS: Twenty-one (0.15%) subjects were diagnosed with Chinese G γ(A γδß)0 -thal. Nine (0.06%) were diagnosed with Southeast Asia HPFH (SEA-HPFH) deletion. Seventy-five (0.53%) cases remained uncharacterized. Three genotypes for Chinese G γ(A γδß)0 -thal and SEA-HPFH deletion were identified, respectively. The genotype-phenotype relationships were discussed. CONCLUSION: Our study for the first time demonstrated that (δß)0 and HPFH were not rare events, and molecular characterized G γ(A γδß)0 -thal and HFPH mutations in the Chinese Zhuang population. The findings in our study will be useful in genetic counseling and prenatal diagnostic service of ß-thalassemia in this populations.

The association between four SNPs (rs7482144, rs4671393, rs28384513 and rs4895441) and fetal hemoglobin levels in Chinese Zhuang ß-thalassemia intermedia patients.

Four SNPs (rs7482144, rs4671393, rs28384513 and rs4895441) associated with HbF levels have been identified in different populations worldwide. To explore whether these SNPs modulate HbF expression in Chinese Zhuang population, 436 Chinese Zhuang ß-thalassemia intermedia (ß-TI) patients were divided into high HbF level group (mean HbF=25.5%, n=218) and low group (mean HbF=6.51%, n=218) for genotyping using PCR-HRM method. Results demonstrated that there was a significantly higher minor allele frequency (MAF=34.2%) of rs4895441 (G) in HMIP in high HbF level group than that in low group (MAF=19.8%) (P=0.001, OR=1.73, 95% CI: 1.24-2.57). The cumulative effects of risk genotypes of these loci for patients carrying any combination of 1, 2 or 3 risk genotype had a gradually increased risk of high HbF level phenotype compared to those without the risk genotypes (OR=1.50-9.06, P=0.0008); Gene-gene interaction of rs7842144 and rs4895441 showed the best model with the smallest prediction error (0.4259) and the greatest consistency of coefficient of variation (P=0.01). We concluded that rs4895441, G on HMIP might be a high-risk modifier variant for high HbF level expression, and HBG2, BCL11A and HMIP genes, as HbF quantitative trait loci (QTL) could have a synergistic effect on increasing the HbF level in Chinese Zhuang ß-TI patients.

A Novel α2-Globin Gene Mutation: Hb Debao [α31(B12)Arg→Trp; HBA2: c.94A>T].

We report a novel mutation on the α2-globin gene, Hb Debao [α31(B12)Arg→Trp; HBA2: c.94A>T] detected in a Chinese family. This mutation gives rise to a previously undescribed hemoglobin (Hb) variant that was undetectable by electrophoretic or chromatographic methods. Hb Debao was associated with an α+-thalassemia (α+-thal) deletion [-α3.7 (rightward)] producing a mild phenotype with significant microcytosis and hypochromia, while the combination of this mutation with an α0-thal deletion (--SEA) resulting in a severe form of Hb H (ß4) disease, which is consistent with a thalassemic phenotype associated with the novel mutation.

A Novel Mutation of the α2-Globin Gene Causing α+-Thalassemia: Hb Nanning (HBA2: c.369_370delinsGA).

We report a novel mutation on the α2-globin gene, Hb Nanning (HBA2:c.369_370delinsGA) detected in a Chinese family. This mutation gives rise to a previously undescribed hemoglobin (Hb) variant that was undetectable by various separation techniques. Both carriers of the mutation have mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) values that are below normal, as would be predicted for an α+-thalassemia (α+-thal) patient.

Characterization of a Large Novel α-Globin Gene Cluster Deletion Causing α0-Thalassemia in a Chinese Family.

We report a large novel α-globin cluster deletion that we named - -PG (NG_000006.1: g.93628_542759del450131), in a Chinese family. This large deletion is approximately 450 kb long, spanning from upstream of the PolR3k gene at the 5' end to the RAB11FIP3 gene at the 3' end of chromosome 16p13.3. This deletion removes all the globin distal regulatory elements as well as the whole α-globin gene cluster. Patients with heterozygous - -PG/αα had red blood cell (RBC) indices consistent with α-thalassemia (α-thal) trait, but no apparent increase in a cancer tendency or mental disability, microcephaly, relative hypertelorism, unusual facies or genital anomalies.

Complex Interaction of Hb Q-Thailand with α0- and ß0-Thalassemia in a Chinese Family.

Hb Q-Thailand [α74(EF3)Asp→His (α1); HBA1: c.223 G>C] is an abnormal hemoglobin (Hb), variant found mainly in China and Southeast Asian countries. The association of the αQ-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report a hitherto undescribed condition of patients with a double heterozygosity for Hb Q-Thailand with α0-thalassemia (α0-thal) and in combination with ß0-thalassemia (ß0-thal) in a Chinese family. Our study will provide some clinical manifestations, laboratory diagnosis and genetic counseling for complex hemoglobinopathies.

Identification of a Novel ß-Globin Mutation (HBB: C.189_195delTCATGGC) in a Chinese Family.

ß-Thalassemia (ß-thal) is one of the most common genetic disorders worldwide. Molecular characterization of ß-thal is essential for prevention and understanding the biology of the disease. More and more rare and novel mutations are being reported. Here, we report a novel 7 bp deletion at codons 63-65 (HBB: c.189_195delTCATGGC) in exon 2 of the ß-globin gene in a family from Guangxi Province, China. This novel mutation causes a shift in the normal reading frame of the ß-globin coding sequence and created a stop codon at codon 87 in exon 2, which leads to a ß(0)-thal phenotype.

First Description of a ß-Thalassemia Mutation, -86 (C > G) (HBB: c.-136C > G), in a Chinese Family.

We present the first description of a Chinese family with a ß-thalassemia (ß-thal) mutation -86 (C > G) (HBB: c.-136C > G). This mutation changes the conserved promoter sequence within the proximal CACCC box of the ß-globin gene that leads to a phenotype of ß(+)-thal. The ß-globin haplotype analysis revealed that the -86 mutation in our case was linked with haplotype I [+ - - - - + +]. This haplotype was commonly found both in the ß-thal mutation and the ß(A) gene. Our results suggest that the -86 mutation possibly does not have a distinct origin.

First Detection of a Splice Site ß-Thalassemia Mutation, IVS-I-6 (T > C) (HBB: c.92 + 6T > C) in a Chinese Family.

We present the first description of Chinese individuals with the ß-thalassemia (ß-thal) mutation IVS-I-6 (T > C) (HBB: c.92 + 6T > C). This mutation interferes with mRNA splicing and results in reducing expression of ß-globin chains that leads to a ß(+)-thal phenotype. The ß-globin haplotype anlaysis revealed the IVS-I-6 mutation in our case was linked with haplotype VI [- + + - - - +] and had Mediterranean characteristics.

HIVID: an efficient method to detect HBV integration using low coverage sequencing.

We reported HIVID (high-throughput Viral Integration Detection), a novel experimental and computational method to detect the location of Hepatitis B Virus (HBV) integration breakpoints in Hepatocellular Carcinoma (HCC) genome. In this method, the fragments with HBV sequence were enriched by a set of HBV probes and then processed to high-throughput sequencing. In order to evaluate the performance of HIVID, we compared the results of HIVID with that of whole genome sequencing method (WGS) in 28 HCC tumors. We detected a total of 246 HBV integration breakpoints in HCC genome, 113 out of which were within 400bp upstream or downstream of 125 breakpoints identified by WGS method, covering 89.3% (125/140) of total breakpoints. The integration was located in the gene TERT, MLL4, and CCNE1. In addition, we discovered 133 novel breakpoints missed by WGS method, with 66.7% (10/15) of validation rate. Our study shows HIVID is a cost-effective methodology with high specificity and sensitivity to identify viral integration in human genome.

A novel variant in NSUN2 causes intellectual disability in a Chinese family.

NSUN2-intellectual disability syndrome, also known as intellectual disability type 5 (MRT5), is an autosomal recessive disorder that is characterized by intellectual disability (ID), postnatal growth retardation, dysmorphic facies, microcephaly, short stature, developmental delay, language impairment and other congenital abnormalities. The disease is caused by mutations in the NSUN2 gene, which encodes a tRNA cytosine methyltransferase that has an important role in spindle assembly during mitosis and chromosome segregation. In this study, we recruited a family that had two individuals with ID. Whole exome sequencing was performed to identify a homozygous frameshift variant (c.1171_1175delACCAT(p.Thr391fs*18*)) in NSUN2 (NM_017755.5) in the proband. The varint was confirmed as segregating in his affected brother and his parents by Sanger sequencing. The individuals that we described showed a similar dysmorphology profile to that associated with MRT5. To analyze the correlations between genotypes of NSUN2 and phenotypes of individuals with ID, we examined 17 variants and the associated phenotypes from 32 ID individuals in current and previous studies. We concluded that mutations in NSUN2 cause a wide range of phenotypic defects. Although some clinical manifestations were highly variable, the core phenotypes associated with NSUN2 mutations were dysmorphic facies, microcephaly, short stature, ID, growth restriction, language impairment, hypotonia and delayed puberty. Our study expands the genetic spectrum of NSUN2 mutations and helps to further define the genotype-phenotype correlations in MRT5.

Compound heterozygous WDR19 variants associated with nephronophthisis, Caroli disease, refractory epilepsy and congenital bilateral central blindness: Case report.

The WDR19 gene has been reported to be involved in nephronophthisis-related ciliopathies such as isolated nephronophthisis 13 (NPHP13), Sensenbrenner syndrome, Jeune syndrome, Senior-Loken syndrome, Caroli disease, retinitis pigmentosa and Asthenoteratospermia. In the present study, we provided the detailed clinical characteristics and genetic analysis of a patient with four variants in WDR19 and TG, reviewed a comprehensive mutation analysis in the WDR19-related ciliopathies, discussed the relationship between genotype and phenotype, and compared the allele frequencies (AFs) of WDR19 variants depending on the ethnic background. We used whole-exome sequencing (WES) combined with bioinformatics analysis to investigate the genetic variants of a 3-year-old boy with common features of WDR19-associated NPHP13 and Caroli disease, bilateral central blindness, refractory epilepsy, and elevated thyroid stimulating hormone. A novel splice-donor variant, c.98+1G > C, and a recurrent missense variant, c.3533G > A, were identified in the WDR19 gene. We used effective mRNA analysis to verify the effects on pre-mRNA processing and to assess the pathogenicity of the splice-site variant. The patient also harbored compound heterozygous variants of the TG gene (c.4889A > G, c.274+2T > G). Of note, using a review of an in-house database, we identified four additional likely pathogenic WDR19 variants and estimated the overall AF of WDR19 mutations to be 0.0025 in the southern Chinese population. Our findings have expanded the allelic spectrum of mutations in the WDR19 gene and broadened the clinical phenotype spectrum of WDR19-related ciliopathies. The results have also provided new insights into the clinical heterogeneity of the disorder, which would be useful in accurate genetic counseling for affected individuals and carrier screening in a general population.