[Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai].

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.

[Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai City from 2015 to 2019].

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai City from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.

[Changes of GSH-PX activity and γ-GCS mRNA expression in serum of workers exposed to manganese].

Objective: To explore the changes of γ-GCS mRNA expression and GSH-PX in serum of workers exposed to manganese in order to provide scientific basis for early diagnosis of manganese poisoning. Methods: In June 2017, a total of 180 workers from a motorcycle manufacturer were selected by stratified random sampling, including 115 welders as the exposure group and 65 administrative office workers as the Control Group, the exposure group was divided into high exposure group (43 persons) and low exposure group (72 persons) according to whether the exposure group exceeded the standard limit. The levels of γ-gcs Mrna expression and GSH-Px activity in serum were determined by Occupational Health Survey, and the differences of γ-gcs Mrna expression and GSH-Px activity among different groups were analyzed. Results: Compared with the control group, the serum GSH-Px activity was lower and the serum γ-GCS mRNA expression level was higher in the exposed group (F=370.52, 275.95, P<0.01) . Compared with the control group, there was significant difference in γ-GCS mRNA expression level and GSH-Px activity (F=0.475、1.06, P<0.01; F=48.53、111.70, P<0.01) . The concentrations of manganese in air, welding dust and urine were positively correlated with the level of γ-GCS mRNA (r=0.71, 0.50, 0.31, P<0.01) The serum GSH-Px activity was negatively correlated with the concentrations of manganese in air, welding dust and urine (r=-0.80, -0.52, -0.30, P< 0.01) , There was no correlation between Serum γ-GSH-Px activity and age and years of exposure (P>0.05) . Conclusion: Serum γ-GCS mRNA expression level and GSH-Px activity level can be used as early biomarkers of manganese poisoning. The concentrations of manganese in workplace air, welding dust and urine manganese in workers are the influencing factors.

Investigation of serum vitamin D levels in Chinese infertile men.

Recently, the question of whether vitamin D exerts an effect on the pathogenic process of infertility has become the centre of attention. There are some controversial conclusions on this issue. Based on previous studies, we sought to explore the difference of serum 25-hydroxyvitamin D3 , 1,25-dihydroxyvitamin D3 levels between infertile patients and fertile men, and to find the influence on semen quality. The analysis of serum 25-hydroxyvitamin D3 level showed no significant difference between infertile patients and fertile men. However, the levels of serum 1,25-dihydroxyvitamin D3 in oligospermia (P < 0.05), asthenospermia (P < 0.01), oligoasthenospermia (P < 0.05) and azoospermia (P < 0.01) patients were significantly lower than those in fertile men. Moreover, serum 1,25-dihydroxyvitamin D3 level was positively correlated with progressive motility and total sperm number in infertile patients. In addition, a positive correlation between serum prolactin and 1,25-dihydroxyvitamin D3 was observed in fertile men. Our results indicated that lower vitamin D could be a risk factor for poor semen quality in infertile men. The 1,25-dihydroxyvitamin D3 , as the biologically active form of vitamin D, may be more significant.

[Exercise-related risk at anaerobic threshold in patients with chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the exercise-related risk at anaerobic threshold(AT) in patients with chronic obstructive pulmonary disease(COPD). METHODS: Sixty two patients [men 56, women 6, aged (66±8) yr] with stable COPD in Beijing Friendship Hospital during 2013-2014, participated in this study. Incremental symptom-limited cardiopulmonary exercise test was performed on cycle ergometer. The AT was determined using the V-Slope technique and ventilatory equivalents for carbon dioxide and oxygen. Symptoms, 10-lead electrocardiogram, oxygen saturation by pulse oximetry(SpO(2)) were monitored during exercise. RESULTS: The AT, detectable in 53 patients, occurred at (68±10)% of peak oxygen uptake(peak VO(2)). The SpO(2) was in the safe range (94±2) % and the respiratory reserve was relatively high at AT (i.e. 48%). CONCLUSIONS: High-intensity exercise training can be performed in patients with moderate-to- severe COPD without resting oxygen desaturation.

First description of NDM-1-, KPC-2-, VIM-2- and IMP-4-producing Klebsiella pneumoniae strains in a single Chinese teaching hospital.

A total of 180 non-duplicate carbapenem-resistant Klebsiella pneumoniae isolates were recovered from patients hospitalized between December 2010 and January 2012 at a Chinese hospital. Eight KPC-2, four NDM-1, one VIM-2, and five KPC-2 plus IMP-4 producers were identified and all were multidrug resistant due to the presence of other resistance determinants, including extended-spectrum ß-lactamases (CTX-M-15, SHV-12), 16S rRNA methylases (armA, rmtB) and plasmid-mediated quinolone-resistance determinants (qnrA, B, S, aac(6')-Ib-cr). Nine K. pneumoniae clones (Kpn-A1/ST395, Kpn-A3/ST11, Kpn-A2/ST134, Kpn-B/ST263, Kpn-C/ST37, Kpn-D/ST39, Kpn-E/ST1151, Kpn-F/ST890, Kpn-G/ST1153) were identified. bla KPC-2 was located on transferable ~65 kb IncL/M (ST395, ST11, ST134, ST39) and ~100 kb IncA/C (ST37, ST1153, ST890) plasmids, respectively. On the other hand, bla NDM-1 was associated with a ~70 kb IncA/C plasmid (ST263). However, non-typable plasmids of ~40 kb containing bla VIM-2 were detected in the ST1151 clone. This work reports the first co-occurrence of four diverse types of carbapenemase of K. pneumoniae clones from a single hospital in China. IncA/C, IncL/M, and other successful plasmids may be important for the dissemination of carbapenemases, producing a complex epidemiological picture.

Correlation of serum IL-6, TNF-α levels and disease activity in patients with ankylosing spondylitis.

OBJECTIVE: The aim of this study was to investigate the serum interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) levels in patients with ankylosing spondylitis (AS), and the correlation between serum levels and disease activity. PATIENTS AND METHODS: ELISA was used to detect the serum TNF-α and IL-6 levels of AS patients (n=40) and normal controls (n=40) who were hospitalized or outpatient-diagnosed from June 2021 to May 2023. C-reactive protein (CRP) was detected by immune-enhanced turbidimetry. The erythrocyte sedimentation rate was determined by Wei's manual method. The correlation was analyzed by Pearson's correlation analysis. RESULTS: The levels of TNF-α and IL-6 in the AS group were significantly higher than those in the control group, and the levels of TNF-α and IL-6 in AS patients in the active phase were higher than those in the stable phase (p<0.05). CRP level was positively correlated with TNF-α, and IL-6 (r=0.02886 and 0.0273, p<0.05). Erythrocyte sedimentation rate (ESR) level was also positively correlated with TNF-α, and IL-6 (r=0.07568 and 0.0613, p<0.05). CONCLUSIONS: Serum TNF-α and IL-6 levels are correlated with AS disease activity, suggesting that they may be involved in the inflammatory response of AS.

[Clinicopathologic characteristics and survival analysis of primary large B-cell lymphoma of the central nervous system].

Objective: To retrospectively analyze the clinical and pathologic characteristics, response to treatment, survival, and prognosis of patients with primary large B-cell lymphoma of the central nervous system (PCNSLBCL) . Methods: Clinical and pathologic data of 70 patients with PCNSLBCL admitted to Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from December 2010 to November 2022 were collected for retrospective analysis. Survival analysis was performed using the Kaplan-Meier method and log-rank test, and prognosis analysis was conducted using the Cox proportional hazards model. Results: Among 70 patients with PCNSLBCL, complete remission (CRs) were achieved in 49 (70.0% ) and partial remission in 4 (5.7% ) after the first-line induction therapy; the overall remission rate was 75.7%. The 2-year progression-free survival (PFS) rate was 55.8% and the median progression-free survival (mPFS) time was 35.9 months, whereas the 2-year overall survival (OS) rate was 79.1% with a median OS time not reached. After CR induced by first-line therapy, cumulative incidence of relapse (CIR) was lower in patients who had received auto-HSCT than in those who had not received consolidation therapy (P=0.032), whose 2-year PFS rate was 54.4% and mPFS time was 35.9 months; comparatively, the 2-year PFS rate in patients having received oral maintenance of small molecule drugs reached 84.4% with a mPFS time of 79.5 months (P=0.038). Multivariant analysis demonstrated that Class 3 in the Memorial Sloan-Kettering Cancer Center (MSKCC) prognostic model is an independent adverse prognostic factor of OS in patients with PCNSLBCL (HR=3.127, 95% CI 1.057-9.253, P=0.039) . Conclusions: In patients with PCNSLBCL achieving CR after the first-line induction therapy, auto-HSCT as consolidation therapy would lead to a decreased CIR, and PFS time could be prolonged by oral maintenance of small molecule drugs. Class 3 MSKCC prognostic model is independently associated with poorer OS.

An elastic-net penalized expectile regression with applications.

To perform variable selection in expectile regression, we introduce the elastic-net penalty into expectile regression and propose an elastic-net penalized expectile regression (ER-EN) model. We then adopt the semismooth Newton coordinate descent (SNCD) algorithm to solve the proposed ER-EN model in high-dimensional settings. The advantages of ER-EN model are illustrated via extensive Monte Carlo simulations. The numerical results show that the ER-EN model outperforms the elastic-net penalized least squares regression (LSR-EN), the elastic-net penalized Huber regression (HR-EN), the elastic-net penalized quantile regression (QR-EN) and conventional expectile regression (ER) in terms of variable selection and predictive ability, especially for asymmetric distributions. We also apply the ER-EN model to two real-world applications: relative location of CT slices on the axial axis and metabolism of tacrolimus (Tac) drug. Empirical results also demonstrate the superiority of the ER-EN model.

LncRNA EBIC promoted proliferation, metastasis and cisplatin resistance of ovarian cancer cells and predicted poor survival in ovarian cancer patients.

OBJECTIVE: Ovarian cancer has the highest mortality rate cancer worldwide in women, and it is the second most common gynecologic malignancy in females, but the treatment remained unsatisfactory. Researches showed that lncRNA EBIC had played key roles in different cancer, but its role in ovarian cancer remains largely unclear. PATIENTS AND METHODS: qRT-PCR was applied to detect the expression of lncRNA EBIC in ovarian cancer and adjacent tissue, and analysis was applied to explore the relationship between expression and clinical characteristic. Overall, the survival curves for the two groups were defined by the high and low expression level of EBIC in ovarian cancer patients. After that, CCK8 and transwell were used to detect the proliferation and metastasis ability of ovarian cancer, after suppression of lncRNA EBIC. The relative protein expression level in ovarian cancer cells after transfection with siRNA-NC or siRNA-EIBC was detected by Western blot. RESULTS: qRT-PCR showed that lncRNA EIBC was highly expressed in ovarian cancer tissue, compared with adjacent tissue. Moreover, we found that expression of lncRNA EIBC was closely related to prognosis, tumor size and lymph node metastasis. We also found that the cell proliferation, invasion, migration and cisplatin resistance in ovarian cancer cells after transfection with siRNA-EBIC were significantly inhibited. Mechanistically, the relative protein expression level of ß-catenin, vimentin and c-myc were significantly decreased and the relative expression of E-cadherin was significantly increased in ovarian cancer cells after transfection with siRNA-EBIC. CONCLUSIONS: We found that overexpression of lncRNA EBIC could promote the proliferation, invasion and migration and improved cells cisplatin resistance by Wnt/ß-catenin signaling pathway in ovarian cancer. LncRNA EBIC may be a potential target for the treatment of ovarian cancer patients.

Inhibition of MMPs Cat G and downregulates the signaling of TGF-ß/Smad in chronic photodamaged human fibroblasts.

OBJECTIVE: To understand the action of Cathepsin G (Cat G) and matrix metalloproteases (MMPs) on the ß/Smad pathway of transforming growth factor ß (TGF-ß) in chronically photodamaged human fibroblasts. Cat G plays a significant role in the process of skin photoaging and in collagen synthesis and degradation which is induced by UV irradiation it could interact with TGF-ß/Smad signaling. No available studies have thoroughly explored its molecular mechanisms of photoaging regulation. PATIENTS AND METHODS: Fibroblasts were divided into 4 groups: (1) control, (2) UVA irradiation of 25 J/cm2, (3) UVA irradiation of 25 J/cm2 + MMPs inhibitor, and (4) 25 J/cm2 UVA irradiation + Cat G inhibitor. All treatments were repeated daily for 21 days. Western blot and ELISA was employed to detect Protein levels for Cat G, MMPs, and several smads. RESULTS: Compared to UVA-irradiated cells, the addition of MMPs inhibitor downregulated the expression of smad2, smad3, and smad4 as well as TGF-ß. The addition of Cat G inhibitor downregulated the expression of smad2, smad3, and smad4 as well as TGF-ß. These data suggest that TGF-ß/Smad signaling was decreased by inhibition of MMPs and Cat G decreased in chronically human fibroblasts which are photo-damaged. CONCLUSIONS: These results may help expand our knowledge of mechanisms mediating photoaging and is possibly instrumental to the exploration of novel anti-photoaging treatments.

Lowering of trichosanthin immunogenicity by site-specific coupling to dextran.

Trichosanthin is a type I ribosome-inactivating protein possessing a broad spectrum of biological and pharmacological activities. Therapeutic use of this compound is hampered by its immunogenicity. It was shown earlier that coupling of dextran to trichosanthin can increase plasma half-life and reduce antigenicity. However, the site where dextran attaches to trichosanthin cannot be controlled; ideally, it should be at or near the antigenic determinant. The present study attempted to couple dextran to trichosanthin at a potential antigenic site. By site-directed mutagenesis, two sites, R29 and K173, were replaced by cysteine, and dextran was coupled to the newly created cysteine residues. The dextran-trichosanthin complex retained 50% of abortifacient activity and had a mean residence time in rats 27-fold longer than natural trichosanthin. Acute hypersensitivity reaction in guinea pigs was reduced greatly after coupling of K173C (a trichosanthin mutant with lysine-173 replaced by cysteine) to dextran. Compared with natural trichosanthin, dextran-K173C had a decrease in IgG and IgE response, whereas the coupling of R29C (a trichosanthin mutant with arginine-29 replaced by cysteine) to dextran did not show significant reduction of immunogenicity. This suggests that K173 but not R29 is located at or near an antigenic determinant. This study has demonstrated an alternative approach for mapping of antigenic determinants. The information obtained is also useful in producing an improved trichosanthin derivative for therapeutic use.

Expression changes of activin A in the development of hepatic fibrosis.

AIM: To examine the expression of activin A, a member of the transforming growth factor (TGFbeta) superfamily, recently has been reported to be overexpressed in liver cirrhosis, in the course of carbon tetrachloride-induced rat hepatic fibrosis. METHODS: Hepatic fibrosis was induced in rats by subcutaneous injections of 40% carbon tetrachloride oily solution for a period of 1 to 7 weeks. At the end of 1, 2, 3, 4, 5, 6 and 7 weeks after carbon tetrachloride injections, the rats were killed in group (6-10 rats each time) for study. The activin A messenger RNA expression and its protein localization were assessed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: The normal rat liver expressed activin A mRNA and protein, and its expression was transiently decreased and became undetectable after carbon tetrachloride injections for 2 or 3 weeks and then increased gradually. After injection of carbon tetrachloride for 6 and 7 weeks, activin A mRNA and protein expressions were significantly enhanced in rat liver. Compared with that of the normal rat liver. Activin A mRNA expression levels in rats receiving carbon tetrachloride injections for 6 and 7 weeks were 1.6 and 2.2 times that of those in normal rat liver respectively (0.456 +/- 0.094 vs 0.2860.0670, P< 0.01; 0.620 +/- 0.134 vs 0.286 +/- 0670, P< 0.01). Immunohistochemistry showed that activin A expressed in hepatocytes of normal liver, and its expression was decreased in rats receiving carbon tetrachloride for 2 or 3 weeks. Compared with normal liver, activin A expression distribution mode changed in fibrotic liver, being increased significantly in hepatocytes around fibrotic areas. CONCLUSION: Activin A expression was increased in late stage of hepatic fibrosis, and this may be involved in hepatic fibrosis formation in this period.

Isolation and molecular analysis of genes Stpk-V2 and Stpk-V3 homologous to powdery mildew resistance gene Stpk-V in a Dasypyrum villosum accession and its derivatives.

Wheat-Dasypyrum villosum translocated chromosomes T6V#2S•6AL and T6V#4S•6DL are known to confer excellent resistance to wheat powdery mildew (PM). However, it is difficult to distinguish the two sources of PM resistance genes through multi-pathotype testing because to date no virulence for them has been found. To reveal the relationship between the PM resistance genes from the two translocations, the sequence of the Stpk-V gene, a key member of powdery mildew resistance locus Pm21, was used as a reference to isolate homologous genes from a D. villosum accession No.1026 and its derivatives 6V#4(6D) disomic substitution (DS) line RW15 and T6V#4S•6DL translocation line Pm97033. Two genes Stpk-V2 and Stpk-V3 were cloned from No.1026. Sequence alignment showed that Stpk-V2 and Stpk-V3 shared 98.2 % and 96.2 % of their DNA and 99.3 % and 100 % of their amino acids in identity with Stpk-V. Compared with Stpk-V, a 22-bp direct sequence repeat and a miniature inverted-repeat transposable element (MITE) were found in the intron 4 of Stpk-V2 and Stpk-V3, respectively. However, Stpk-V2 was not present in DS line RW15 and translocation line Pm97033 based on the PCR result, indicating that Stpk-V2 did not contribute to the PM resistance of RW15 and Pm97033. In the promoter region, a 78-bp insertion was found not only in Stpk-V2 and Stpk-V3, but also in its orthologous gene Stpk-A of wheat. In addition, there was a 17 bp/8 bp deletion/insertion in the putative promoter of Stpk-V3 in comparison with that of Stpk-V/Stpk-V2. Real-time quantitative RT-PCR analysis indicated that the expression levels of Stpk-V and Stpk-V3 genes in the translocation lines were induced by the pathogen, but Stpk-V had a higher expression level than Stpk-V3 at 12 h after inoculation with Bgt. The diversity of Stpk-V gene will help to explore new resistance genes to PM in D. villosum for wheat breeding.

Retrieval of atmospheric optical parameters from airborne flux measurements: application to the atmospheric correction of imagery.

Methodologies that employ auxilliary flux data collected by upward- and downward-looking optical sensors to improve atmospheric corrections of airborne multispectral images are presented and evaluated. Such flux data often are collected in current airborne sensors to produce bidirectional reflectance factor (BRF) images and estimates of hemispherical-hemispherical reflectance. The fact that these images must then be corrected for atmospheric interference raises the question as to whether the auxilliary flux information can be employed to estimate some of the input parameters required by atmospheric correction models. Radiative transfer simulations are employed to demonstrate that the utilization of the downwelling and upwelling fluxes as a means of inferring intrinsic atmospheric optical information can be used to better characterize the local atmosphere and accordingly to improve the atmospheric corrections applied to the apparent BRF images.

Structure/function relationship study of Tyr14 and Arg22 in trichosanthin, a ribosome-inactivating protein.

Amino acids Tyr14 and Arg22 in trichosanthin are residues on helix A1 close to the active-site cleft. They are invariant in various type-I and type-II ribosome-inactivating proteins. In this study, Tyr14 was changed to Phe and Arg22 to Lys and Leu. Modified proteins were purified, and activities compared by assaying their median inhibitory concentration (ID50) on a rabbit-reticulocyte-lysate protein-synthesis system. While the ID50 of wild-type trichosanthin was 0.02 nM, those for [Phe14], [Lys22], [Leu22] and [Phe14, Leu22]trichosanthin were 0.10, 0.03, 0.25 and 0.15 nM, respectively. Therefore, compared with Tyr14, Arg22 appears to play a more important role in trichosanthin. Structural studies on [Leu22]trichosanthin showed that two water molecules occupy the space left by the side chain of Arg22, and hydrogen bonds exist between these water molecules and nearby residues to retain the conformation. The use of intermolecular rather than intramolecular hydrogen bonds may have an adverse effect on stability or folding of the protein and results in a mild decrease in activity.