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1.
Mol Med Rep ; 22(5): 3667-3676, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33000231

RESUMO

Recent studies have revealed that long non­coding RNAs (lncRNAs) serve important roles in carcinogenesis and that this type of gene may be used as biomarkers in cancer. A high level of lncRNA HOXA distal transcript antisense RNA (HOTTIP) is associated with unfavorable prognosis for patients with ovarian cancer (OC), but the mechanism of HOTTIP involved in OC development remains to be elucidated. The present study aimed to investigate the mechanism of HOTTIP in metastasis­associated OC cell behaviors. HOTTIP levels in ovarian cells were quantified by reverse transcription­quantitative PCR, cell proliferation was analyzed by colony formation assay, and apoptosis was assessed by flow cytometry. Cell migratory and invasive abilities were evaluated by wound healing and Transwell assays, respectively. The expression levels of mitogen­activated protein kinase kinase (MEK)/ERK pathway­associated proteins were detected by western blotting. The results demonstrated that knockdown of HOTTIP in OC cells significantly reduced the phosphorylation levels of MEK and ERK, inhibited the proliferation and invasion of OC cells and promoted their apoptosis. Furthermore, the effects of HOTTIP on cell migration and invasion were partly associated with the epithelial­mesenchymal transition (EMT) process. Proliferation, invasion and EMT of OC cells were enhanced following overexpression of HOTTIP; however, these effects were reversed by the MEK/ERK pathway inhibitor U0126. In conclusion, HOTTIP was demonstrated to promote the proliferation, migration and invasion of OC cells by activating the MEK/ERK pathway. Therefore, HOTTIP may serve as a potential therapeutic target for OC.

2.
J Agric Food Chem ; 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33030899

RESUMO

Here, we first developed a combined strain improvement strategy of biosensor-guided atmospheric and room-temperature plasma mutagenesis and genome shuffling. Application of this strategy resulted in a 2.7-fold increase in the production of shikimic acid (SA) and a 2.0-fold increase in growth relative to those of the starting strain. Whole-cell resequencing of the shuffled strain and confirmation using CRISPRa/CRISPRi revealed that some membrane protein-related mutant genes are identified as being closely related to the higher SA titer. The engineered shuffling strain produced 18.58 ± 0.56 g/L SA from glucose with a yield of 68% (mol/mol) by fed-batch whole-cell biocatalysis, achieving 79% of the theoretical maximum. Sucrose-utilizing Escherichia coli was engineered for SA production by introducing Mannheimia succiniciproducens ß-fructofuranosidase gene. The resulting sucrose-utilizing E. coli strain produced 24.64 ± 0.32 g/L SA from sucrose with a yield of 1.42 mol/mol by fed-batch whole-cell biocatalysis, achieving 83% of the theoretical maximum.

3.
J Am Acad Dermatol ; 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33031838

RESUMO

BACKGROUND: Observational studies have shown promising therapeutic effects of long-pulsed neodymium-doped yttrium aluminum garnet (LP-Nd:YAG) laser on warts. OBJECTIVE: To evaluate whether LP-Nd:YAG laser was superior to cryotherapy for cutaneous warts. METHODS: In this study, 150 adult patients with warts were randomized equally to receive laser or cryotherapy every 3-4 weeks, a maximum of 4 sessions. The primary outcomes were cure rate at 16 weeks and 6 months; secondary outcomes included time to clearance of warts and treatment-related adverse effects. RESULTS: There was no difference in the cure rate for laser versus cryotherapy at 16 weeks (54.1% vs. 46.7%) and 6 months (59.5% vs. 57.3%). However, time to clearance of warts, up to 16 weeks and 6 months, tended to be shorter for laser versus cryotherapy ( P = .04 and .08, respectively). Post-hoc analyses showed a significantly higher cure rate for laser versus cryotherapy in 3 subgroups of HPV 2/27/57-induced recalcitrant warts, but not in their counterpart subgroups. Laser had more mild adverse effects. LIMITATIONS: Single-center. CONCLUSIONS: The overall therapeutic effects of LP-Nd:YAG laser were similar to cryotherapy, but laser may be more effective to relatively recalcitrant warts and may associate with shorter time to clearance of warts.

4.
Kaohsiung J Med Sci ; 2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33022894

RESUMO

Cullin 4B (CUL4B) was reported to be closely related to the progression of some tumors, but its function in clear cell renal cell carcinoma (ccRCC) has not been reported. Our present study found CUL4B was upregulated in ccRCC, and CUL4B knockdown markedly inhibited ccRCC cell growth and induced apoptosis. In addition, CUL4B knockdown markedly inhibited antiapoptotic proteins' expression in ccRCC cells, including Mcl-1 and Bcl-2, and silenced CUL4B also induced the cleavages of PARP, an important index of apoptosis. We also confirmed microRNA-217 (miR-217) was downregulated in ccRCC tumor tissues, and negatively correlated with CUL4B expression. Further investigations revealed miR-217 targeted CUL4B and markedly inhibited its expression in ccRCC cells. In addition, overexpression of miR-217 by mimics significantly suppressed ccRCC cell growth. In contrast, enforced expression of CUL4B significantly abolished miR-217-induced cell survival inhibition in ccRCC cells. In conclusion, our present results suggested targeting miR-217-CUL4B axis would be a promising strategy for ccRCC treatment.

5.
Artigo em Inglês | MEDLINE | ID: mdl-33023290

RESUMO

For hole-conductor-free, fully printable mesoscopic perovskite solar cells (MPSCs), it is difficult to achieve free and efficient diffusion of perovskite precursors in micron-scale porous structures. Thus, the wettability of the perovskite precursor is one of the most crucial factors that determine the performance of MPSCs. Here, d-sorbitol hexaacetate (DSHA) is introduced as an additive for fabricating hole-conductor-free, fully printable MPSCs based on methylammonium lead iodide (MAPbI3). The fabricated MPSCs exhibited an efficiency of 14.33%. Moreover, the influence of DSHA on the optical properties, morphology, and filling of perovskite in the MPSCs has been systematically investigated. The results revealed that DSHA effectively optimized the morphology, improved the pore-filling, and passivated the defects of perovskite films. Remarkably, the unencapsulated MPSCs retained 93% of their original power-conversion efficiency (PCE) after 45 days of storage in air with humidity of 50 ± 5%.

6.
Sci Signal ; 13(652)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33023986

RESUMO

Differences in the relative abundances of the progesterone receptor (PGR) isoforms PGRA and PGRB are often observed in women with reproductive tract cancers. To assess the importance of the PGR isoform ratio in the maintenance of the reproductive tract, we generated mice that overexpress PGRA or PGRB in all PGR-positive tissues. Whereas few PGRA-overexpressing mice developed reproductive tract tumors, all PGRB-overexpressing mice developed ovarian neoplasms that were derived from ovarian luteal cells. Transcriptomic analyses of the ovarian tumors from PGRB-overexpressing mice revealed enhanced AKT signaling and a gene expression signature similar to those of human ovarian and endometrial cancers. Treating PGRB-overexpressing mice with the PGR antagonist RU486 stalled tumor growth and decreased the expression of cell cycle-associated genes, indicating that tumor growth and cell proliferation were hormone dependent in addition to being isoform dependent. Analysis of the PGRB cistrome identified binding events at genes encoding proteins that are critical regulators of mitotic phase entry. This work suggests a mechanism whereby an increase in the abundance of PGRB relative to that of PGRA drives neoplasia in vivo by stimulating cell cycling.

7.
Chem Asian J ; 2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33025769

RESUMO

An unprecedented copper-catalyzed heteroaromatization/sulfonyl transfer of propargylic alcohols with isocyanide has been developed. 3-Sulfonyl benzofurans and indoles were produced under Cu(I) catalysis in good to high yields. The developed catalytic methodology provides controlled, modular, and facile access to sulfonyl benzoheterocycle scaffolds.

8.
Chem Commun (Camb) ; 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33026408

RESUMO

A new hybrid organic-inorganic film, tincone, was developed by using molecular layer deposition (MLD), and exhibited high electrochemical activity toward Li storage. The self-limiting growth behavior, high uniformity on various substrates and good Li-storage performance make tincone a very promising new anode material for 3D microbatteries.

9.
Org Biomol Chem ; 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33026409

RESUMO

Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of glucosamine to synthesize HS with desired biological activities. Chemoenzymatic synthesis is a widely adopted method to obtain HS oligosaccharides to support biological studies. However, this method is unable to synthesize all possible structures due to the specificity of natural enzymes. Here, we report the use of an engineered 6-OST to achieve fine control of the 6-O-sulfation. Unlike wild type enzyme, the engineered 6-OST only sulfates the non-reducing end glucosamine residue. Utilizing the engineered enzyme and wild type enzyme, we successfully completed the synthesis of five hexasaccharides and one octasaccharide differing in 6-O-sulfation patterns. We also identified a hexasaccharide construct as a new anticoagulant drug candidate. Our results demonstrate the feasibility of using an engineered HS biosynthetic enzyme to prepare HS-based therapeutics.

10.
Circ Cardiovasc Interv ; : CIRCINTERVENTIONS120009155, 2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33040580

RESUMO

BACKGROUND: The potential impact of quantitative flow ratio (QFR) based functional Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery (SYNTAX) score (FSSQFR) on prognostication and revascularization strategy choice has not been fully investigated, and the discriminant ability of FSSQFR needs further validation. METHODS: QFR was retrospectively analyzed in left main or patients with multivessel coronary artery disease from the PANDA III trial. A total of 607 patients with analyzable QFR in all vessels were included. FSSQFR was counted by summing the individual scores only in ischemia-producing lesions (vessel QFR ≤0.8). Patients were stratified according to tertiles of SYNTAX score (SS), and 3 groups of FSS were divided by the same cutoff score. The primary end point was 2-year major adverse cardiac events (a composite of cardiac death, any myocardial infarction, or ischemia-driven revascularization). RESULTS: After calculating the FSSQFR, 16% (96/607) of study patients moved from higher-risk group by SS to lower-risk group. In the low, intermediate, and high FSSQFR group, the cumulative incidence of 2-year major adverse cardiac events was 9.1%, 13.5%, and 22.3% (P=0.0004), and the rate of a composite of cardiac death or myocardial infarction (3.8%, 7.3%, and 13.7%, P=0.0006) was also increased. Compared with SS, FSSQFR significantly improved risk classification and prognostication (area under the curve of the receiver-operating characteristics 0.65 versus 0.62, P=0.0009). Moreover, 6% (38/607) of patients, for whom coronary artery bypass grafting would be recommended according to SS, converted to favor percutaneous coronary intervention after FSSQFR calculation. After multivariate adjustment, FSSQFR was an independent predictor of 2-year major adverse cardiac events (adjusted hazard ratio, 1.05 [95% CI, 1.02-1.07]; P=0.0001). CONCLUSIONS: Among patients with left main or multivessel coronary artery disease, FSSQFR showed applicability in prognostication and revascularization strategy choice. An improved scoring system combining anatomy and physiology (FSSQFR) discriminated the risk of adverse events modestly better than anatomic assessment (SS) alone. REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02017275.

11.
Small ; : e2004557, 2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33043568

RESUMO

Realizing near-infrared (NIR) driven photocatalytic reaction is one of the promising strategies to promote the solar energy utilization and photocatalytic efficiencies. However, effective reactive oxygen species (ROS) activation under NIR irradiation remains to be great challenge for nearly all previously reported photocatalysts. Herein, the cubic-phase tungsten nitride (WN) with strong plasmonic NIR absorption and low-work function (≈3.59 eV) is proved to be able to mediate direct ROS activation by both of experimental observation and theoretical simulation. The cubic WN nanocubes (NCs) are synthesized via the hydrothermal-ammonia nitridation process and its NIR-driven photocatalytic properties, including photocatalytic degradation, hydroxylation, and de-esterification, are reported for the first time in this work. The 3D finite element simulation results demonstrate the size dependent and wavelength tuned plasmonic NIR absorption of the WN NCs. The NIR-driven photocatalytic mechanism of WN NCs is proposed based on density functional theory (DFT) calculated electronic structure and facet dependent O2 (or H2 O) molecular activation, radicals scavenging test, spin trapped electron paramagnetic resonance measurements, and ultraviolet photoelectronic spectrum (UPS). Overall, the results in this work pave a way for the application of low-work-function materials as highly reactive NIR photocatalyst.

12.
Artigo em Inglês | MEDLINE | ID: mdl-33036990

RESUMO

Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain to be an important task in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum into producing ethanol, we attained a low yield (63% of the theoretical), when using resting cells on glucose, and large amounts of succinate and acetate were formed. To prevent the by-products formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with the one from Escherichia coli, which is only active under aerobic conditions. However, this tampering resulted in a 10 times reduced glycolytic flux as well as a greatly increased NADH/NAD+ ratio. By substituting glucose with fructose, we found that the glycolytic flux was greatly enhanced, which led us to speculate whether the source of reducing power could be the the pentose phosphate pathway (PPP) that is bypassed when fructose is metabolized. Indeed, after shutting down the PPP by deleting the zwf gene, encoding glucose-6-phosphate dehydrogenase, the ethanol yield on glucose increased significantly to 92% of the theoretical. Based on that, we managed to re-channel the metabolism of C. glutamicum into D-lactate with high yield (98%), which is the highest that has been reported. It is further demonstrated that the PPP-inactivated plaform strain can offer high-yield production of valuable chemicals using lactose contained in dairy waste as feedstock, which paves a promising way for potentially turning dairy waste into value.Importance The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under non-growing conditions and we demonstrate here that the PPP in resting C. glutamicum is a source of reducing power that can interfere with otherwise redox balanced metabolic pathways and reduce yields of desired products. By harnessing this physiological insight, we employed the PPP-inactivated platform strains to produce ethanol, D-lactate and alanine using the dairy waste - whey permeate as the feedstock. The production yield is high and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use non-growing C. glutamicum cells for producing valuable compounds. Overall, we described the benefits to disrupt the oxidative PPP in non-growing C. glutamicum and provide a feasible approach towards waste valorization.

13.
Innovation (N Y) ; : 100046, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-33016958

RESUMO

[This corrects the article DOI: 10.1016/j.xinn.2020.100028.].

14.
Inflammopharmacology ; 28(6): 1481-1493, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33006110

RESUMO

Gout, the most prevalent inflammatory arthritis worldwide, released interleukin-1ß (IL-1ß) and Cathepsin B inflammatory mediators that constitute the hallmark of the disease. Herein we aimed to investigate whether procyanidin B2 (PCB2), a natural dietary compound, can suppress MSU crystals-stimulated gouty inflammation. Treated with lipopolysaccharide (LPS) plus MSU, both mouse peritoneal macrophages (MPM) and mouse bone marrow-derived macrophages (BMDM) released a large amount of mature IL-1ß compared to those treated with MSU or LPS alone, while IL-1ß release was blocked by TLR4 and its downstream effector inhibitors. In two mouse models of gout, oral administration of PCB2 suppressed MSU crystals-induced increasing expression of IL-1ß, Cathepsin B and NLRP3 in the air pouch skin and paws, accompanied with the downregulation prostaglandin E2 (PGE2) in pouch exudates. Inflammatory immune cell infiltration including macrophages and neutrophils were significantly blocked by PCB2 in air pouch skin and paws of mice gout groups. PCB2 also suppressed the release of IL-1ß and Cathepsin B induced by MSU plus LPS in MPM. Our results suggest that the inhibitory effects of PCB2 on NLRP3 inflammasome may alleviate inflammatory response in gout, and this might be a promising anti-inflammatory mechanism of PCB2 against the inflammation in gout.

15.
Chemosphere ; 264(Pt 1): 128450, 2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-33007573

RESUMO

BPA analogs, including bisphenol S (BPS) and bisphenol B (BPB), have been used to replace BPA since it was banned to be added. To investigate whether BPA and its analogs cause oxidative damage effects on primary hippocampal neurons of rats, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), mitochondrial membrane potential (MMP), apoptosis and cell viability assays were conducted after hippocampal neurons exposure to different concentrations of BPA, BPS, and BPB (1, 10, 100 nM and 1, 10, 100 µM). Moreover, the effects of EGCG (5 and 6 µM for male and female, respectively) added on neurons exposed to BPA were assessed. Results showed that 24 h exposure to these bisphenols (BPs) could increase the levels of ROS and contents of MDA, but reduce the activity of SOD significantly. A decline of cell viabilities accompanied with the increasing of apoptosis rates was observed after 7 d exposure to BPs and the reduction of MMP was also observed after 7 d exposure to BPA. Interestingly, BPS has the lower toxicity to hippocampal neurons compared with BPA and BPB. Non-monotonic dose-effect relationships between the concentrations of BPs and the cytotoxic effects were observed, and the effects of BPs on male hippocampal neurons are greater than those of female ones in general. While EGCG can protect neurons free of oxidative damages. In conclusion, the results suggest that BPs may induce sex-specific neurotoxic effects involving oxidative stress, which can be attenuated by EGCG, and males are more sensitive to BPs than females.

16.
Neural Netw ; 132: 461-476, 2020 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-33039785

RESUMO

Generative adversarial networks have been extensively studied in recent years and powered a wide range of applications, ranging from image generation, image-to-image translation, to text-to-image generation, and visual recognition. These methods typically model the mapping from latent space to image with single or multiple generators. However, they have obvious drawbacks: (i) ignoring the multi-modal structure of images, and (ii) lacking model interpretability. Importantly, the existing methods mostly assume one or more generators can cover all image modes even if we do not know the structure of data. Thus, mode dropping and collapse often take place along with GANs training. Despite the importance of exploring the data structure in generation, it has been almost unexplored. In this work, aiming at generating multi-modal images and interpreting model explicitly, we explore the theory on how to integrate GANs with data structure prior, and propose latent Dirichlet allocation based generative adversarial networks (LDAGAN). This framework is extended to combine with a variety of state-of-the-art single-generator GANs and achieves improved performance. Extensive experiments on synthetic and real datasets demonstrate the efficacy of LDAGAN for multi-modal image generation. An implementation of LDAGAN is available at https://github.com/Sumching/LDAGAN.

17.
BMC Complement Med Ther ; 20(1): 304, 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33032580

RESUMO

BACKGROUND: Hypertension, a major risk factor of cardiovascular mortality, is a critical issue for public health. Although Baduanjin (Eight Brocades, EB), a traditional Chinese exercise, might influence blood pressure, glucose, and lipid status, the magnitude of true effects and subgroup differences remains unclear. Therefore, we performed a systematic review of relevant randomized controlled trials (RCTs) to evaluate the effect of EB on patient-important outcomes. METHODS: We systematically searched PubMed, the Cochrane Library, Web of Science, and Chinese databases since inception until March 30, 2020. Meta-analysis was carried out using "meta" package in R 3.4.3 software. A prespecified subgroup analysis was done according to the type of comparisons between groups, and the credibility of significant subgroup effects (P < 0.05) were accessed using a five-criteria list. A GRADE evidence profile was constructed to illustrate the certainty of evidence. RESULTS: Our meta-analysis, including 14 eligible trials with 1058 patients, showed that compared with routine treatment or health education as control groups, the mean difference (MD) in systolic blood pressure (SBP) of the EB groups was - 8.52 mmHg (95%CI:[- 10.65, - 6.40], P < 0.01) and diastolic blood pressure (DBP) was - 4.65 mmHg (95%CI: [- 6.55, - 2.74], P < 0.01). For blood pressure, the evidence was, however, of low certainty because of risk of bias and inconsistency, and for the outcomes of most interest to patients (cardiovascular morbidity and mortality directly), of very low certainty (measurement of surrogate only). Subgroup analysis showed there was no significant interaction effect between different type of comparisons (SBP P = 0.15; DBP P = 0.37), so it could be easily attributed to chance. CONCLUSION: Regularly EB exercising may be helpful to control blood pressure, but the evidence is only low certainty for blood pressure and very low certainty for cardiovascular morbidity and mortality. Rigorously designed RCTs that carry out longer follow-up and address patient-important outcomes remain warranted. TRIAL REGISTRATION: PROSPERO Registration number: CRD42018095854 .

18.
Arch Biochem Biophys ; 695: 108623, 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33039388

RESUMO

Renal cell carcinoma (RCC) is a frequently diagnosed cancer with high prevalence, which is inversely associated with survival benefit. Although myriad studies have shed light on disease causality, unfortunately, thus far, RCC diagnosis is faced with numerous obstacles partly due to the insufficient knowledge of effective biomarkers, hinting deeper mechanistic understanding are urgently needed. Metabolites are recognized as final proxies for gene-environment interactions and physiological homeostasis as they reflect dynamic processes that are ongoing or have been taken place, and metabolomics may therefore offer a far more productive and cost-effective route to disease discovery, particularly within the arena for new biomarker identification. In this review, we primarily expatiate recent advances in metabolomics that may be amenable to novel biomarkers or therapeutic targets for RCC, which may expand our armaments to win more bettles against RCC.

19.
Curr Opin Cell Biol ; 68: 45-54, 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33039945

RESUMO

Cortical actin waves have emerged as a widely prevalent phenomena and brought pattern formation to many fields of cell biology. Cortical excitabilities, reminiscent of the electric excitability in neurons, are likely fundamental property of the cell cortex. Although they have been mostly considered to be biochemical in nature, accumulating evidence support the role of mechanics in the pattern formation process. Both pattern formation and mechanobiology approach biological phenomena at the collective level, either by looking at the mesoscale dynamical behavior of molecular networks or by using collective physical properties to characterize biological systems. As such they are very different from the traditional reductionist, bottom-up view of biology, which brings new challenges and potential opportunities. In this essay, we aim to provide our perspectives on what the proposed mechanochemical feedbacks are and open questions regarding their role in cortical excitable and oscillatory dynamics.

20.
BMC Gastroenterol ; 20(1): 334, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33054747

RESUMO

BACKGROUND: We report a rare case of primary abdominal cocoon with bilateral cryptorchidism. CASE PRESENTATION: The patient had a history of laparoscopic surgery for bilateral cryptorchidism 6 years earlier. He was admitted to the hospital again due to intestinal obstruction. Surgery was performed on the patient after the failure of conservative treatment. The patient was diagnosed with primary abdominal cocoon. Instead of the greater omentum, many cocoon-like tissues surrounding the bowel were seen during operation. Abdominal surgery can increase the risk of intestinal adhesion, which is one of the main causes of intestinal obstruction, especially in patients with abdominal cocoon. We hypothesize that the surgery 6 years earlier to address transabdominal bilateral cryptorchidism accelerated the patient's intestinal obstruction. CONCLUSION: This case implies that it is important for urologists to evaluate whether their patients exhibit abdominal cocoon before cryptorchidism surgery, to choose better surgical methods and reduce the risks of poor prognosis.

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