Preliminary application of foldable pedicled latissimus dorsi myocutaneous flap for repairing soft tissue defects in shoulder and back / 中国修复重建外科杂志

OBJECTIVE@#To explore the feasibility and effectiveness of a foldable pedicled latissimus dorsi myocutaneous flap to repair soft tissue defects in the shoulder and back.@*METHODS@#Between August 2018 and January 2023, the foldable pedicled latissimus dorsi myocutaneous flaps were used to repair soft tissue defects in the shoulder and back of 8 patients. There were 5 males and 3 females with the age ranged from 21 to 56 years (mean, 35.4 years). Wounds were located in the shoulder in 2 cases and in the shoulder and back in 6 cases. The causes of injury were chronic infection of skin and bone exposure in 2 cases, secondary wound after extensive resection of skin and soft tissue tumor in 4 cases, and wound formation caused by traffic accident in 2 cases. Skin defect areas ranged from 14 cm×13 cm to 20 cm×16 cm. The disease duration ranged from 12 days to 1 year (median, 6.6 months). A pedicled latissimus dorsi myocutaneous flap was designed and harvested. The flap was divided into A/B flap and then were folded to repair the wound, with the donor area of the flap being pulled and sutured in one stage.@*RESULTS@#All 7 flaps survived, with primary wound healing. One patient suffered from distal flap necrosis and delayed healing was achieved after dressing change. The incisions of all donor sites healed by first intention. All patients were followed up 6 months to 4 years (mean, 24.7 months). The skin flap has a good appearance with no swelling in the pedicle. At last follow-up, 6 patients had no significant difference in bilateral shoulder joint motion, and 2 patients had a slight decrease in abduction range of motion compared with the healthy side. The patients' daily life were not affected, and linear scar was left in the donor site.@*CONCLUSION@#The foldable pedicled latissimus dorsi myocutaneous flap is an ideal method to repair the soft tissue defect of shoulder and back with simple operation, less damage to the donor site, and quick recovery after operation.

SARS-CoV-2 spike host cell surface exposure promoted by a COPI sorting inhibitor

Via an insufficient coat protein complex I (COPI) retrieval signal, the majority of SARS-CoV-2 spike (S) is resident in host early secretory organelles and a tiny amount is leaked out in cell surface. Only surface-exposed S can be recognized by B cell receptor (BCR) or anti-S therapeutic monoclonal antibodies (mAbs) that is the trigger step for B cell activation after S mRNA vaccination or infected cell clearance by S mAbs. Now, a drug strategy to promote S host surface exposure is absent. Here, we first combined structural and biochemical analysis to characterize S COPI sorting signals. A potent S COPI sorting inhibitor was then invented, evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity (ADCC). Importantly, with the inhibitor as a probe, we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations, possibly corresponding to its ER chaperone association. Our findings not only suggest COPI is a druggable target against COVID-19, but also highlight SARS-CoV-2 evolution mechanism driven by S folding and trafficking mutations.

Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer

Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE + DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both in vitro and in vivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE + DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.

Specific blockade of muscle acetylcholine receptor by α-conotoxin MIA and MIB / 药学学报

The α-conotoxins are peptide toxins that are identified from the venom of marine cone snails and they hold outstanding potency on various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, so nAChR dysfunctions have been involved in a variety of severe pathologies. Four types of α-3/5 conotoxins MI, MIA, MIB and MIC have been found from Conus magus. Among them, the activity and selectivity of MIA and MIB have not been well studied. In this study, four α-3/5 conotoxins MI, MIA, MIB and MIC were synthesized by solid peptide synthesis method, and the bioactivities of them were screened by double electrode voltage clamp electrophysiology. The results showed that MIA and MIB selectively inhibited muscle type acetylcholine receptors with IC50 values of 14.45 and 72.78 nmol·L-1, respectively, which are slightly weaker than MI and MIC. Molecular docking results have shown MIA and MIB interact with muscle-type nAChRs with similar mechanism. The reasons for activity differences may relate to the size of the N-terminal amino acids. Together, the conotoxins MIA and MIB may have the potential to develop as a tool for detect the function of muscle type nAChRs, as well as the diagnosis or treat of related diseases.

Structure and Function of Glycosyltransferase from Common Pathogenic Bacteria / 中国生物化学与分子生物学报

Glycosyltransferases (GTs) catalyze the transfer of sugar moieties from activated donormolecules to acceptors such as sugars, lipids, proteins, and nucleic acids. Protein glycosylation is one ofthe most important post-translational modifications (PTMs). In recent years, increasing studies haveshown that glycosyltransferases are closely related to the virulence of pathogenic bacteria, and play a keyrole in adhesion, immune evasion, and host colonization. According to the features of three-dimensionalstructure, glycosyltransferases are classified into three groups (GT-A, GT-B and GT-C), among whichGT-A and GT-B folds are more common. Glycosyltransferases, which play a role in bacterial adhesion, adopt the GT-B or GT-C fold and glycosylate the surface proteins of pathogenic bacteria (adhesionproteins, autotransporters, etc.). It plays an important role in the adhesion of pathogenic bacteria, theformation of biofilm, and the virulence mechanisms. Glycosyltransferases take part in bacterial adhesionprocess of infection, and glycosyltransferases belonging to GT-A directly glycosylate host proteins andaffect host signal transduction, protein translation, and immune response. This review discusses thestructure of common pathogenic bacteria glycosyltransferases and the pathogenic mechanisms underlyingthese diseases of glycosylation. One kind of glycosyltransferases mainly modify their surface proteins, suchas the glycosyltransferase for specifically glycosylating high-molecular-weight(HMW) adhesion proteins, glycosyltransferases for glycosylation modification of serine-rich repeat proteins (SRRPs), bacterialautotransporter heptosyltransferase (BAHT) family, and N-linked protein system. The other kinds ofglycosyltransferases modulate host responses by directly modifying host proteins, such as Clostridium largecytotoxin, Legionella glycosyltransferase, and the NleB effector from enterobacteria. This review providesa reference for systematically revealing the pathogenic mechanism of glycosyltransferase in pathogenicbacteria, and contributes scientific knowledge in the development of pathogenic bacteria diagnosis, drugdesign, and vaccine development.

Amiloidosis AL: conceptos actuales / AL amyloidosis: current concepts

Las amiloidosis sistémicas constituyen un grupo de enfermedades con diversas etiologías, caracterizadas por la síntesis de proteínas con plegado defectuoso, capaces de agregarse y depositarse en el medio extracelular de diferentes órganos y tejidos, alterando su estructura y función. Se conocen más de 14 formas de amiloidosis sistémica, de las cuales la más frecuente es la amiloidosis AL, objeto de esta revisión, en la que las proteínas precursoras son cadenas ligeras de inmunoglobulina inestables, secretadas por un clon de células plasmáticas o, con menor frecuencia, por un linfoma linfoplasmocítico o de células del manto. La amiloidosis AL puede llevar a una amplia gama de manifestaciones clínicas y compromiso de órganos, como el corazón y el riñón. El reconocimiento temprano de la enfermedad y el diagnóstico oportuno son determinantes para mejorar la supervivencia de los pacientes. El tratamiento deberá ser individualizado de acuerdo con la condición de cada paciente, lo que hace necesaria una correcta clasificación de los individuos según su pronóstico. La terapia dirigida a la amiloidosis está enfocada esencialmente en disminuir el compromiso orgánico, y por ende, prolongar la supervivencia con mejoría en los síntomas. En esta revisión se discutirán aspectos importantes de la fisiopatología, epidemiología, manifestaciones clínicas, diagnóstico y tratamiento de la amiloidosis AL

Systemic amyloidosis constitutes a group of diseases with diverse etiologies characterized by the synthesis of proteins with defective folding, capable of aggregating and depositing in the extracellular matrix of different organs and tissues, altering their structure and function. More than 14 forms of systemic amyloidosis are known, of which the most frequent is AL amyloidosis, the subject of this review, in which the precursor proteins are unstable immunoglobulin light chains, secreted by a clone of plasma cells or, to a lesser extent, often due to lymphoplasmacytic or mantle cell lymphoma. AL amyloidosis can lead to a wide range of clinical manifestations and organ involvement, such as the heart and kidney. Early recognition of the disease and timely diagnosis are crucial to improve patient survival. Treatment should be individualized according to the condition of each patient, which requires a properly classification of individuals according to their prognosis. Amyloidosis-targeted therapy is essentially focused on reducing organ involvement, and therefore prolonging survival with improvement in symptoms. In this review, important aspects of the pathophysiology, epidemiology, clinical manifestations, diagnosis, and treatment of AL amyloidosis are discussed

Modelado de estructuras-3D de mutaciones raras del receptor-1-melanocortina asociadas al melanismo en el mielero / Modelling of 3d-structures of the rare melanocortin-1-receptor mutations associated to melanism in the bananaquit

ABSTRACT Melanism in plumage color is often associated to the single nucleotide polymorphism of the melanocortin-1-receptor. Despite the striking association between the substitution of a Glutamic-acid by for a Lysine at position 92 on the MC1R protein and a completely black plumage, an in-depth understanding of the effect of missense mutations on the conformational change and behavior of the MC1R in the lipid bilayer caused by the absence of a crystal structure is lacking. We examine the structural basis for receptor activation using DNA sequences from the GenBank to perform in silico protein homology-based modeling. Our tridimensional model shows that the Alanine for a 179-Threonine substitution is a structural complement of the charge-reversing effect associated to the substitution of a Glutamic-acid by for a Lysine at position 92 on the MC1R. We proposed the possibility of gradual evolution in stability and electrostatic properties of the MC1R by the sequential accumulation of these two rare substitutions. These two rare substitutions further perturb physical-chemical properties that may be necessary folding requirements of the constitutively active MC1R forms without altering of ligand binding affinity. The computational coarse-grained molecular dynamics of the MC1R binding affinities to the melanocyte-stimulating hormone predicted the disparity in ligand binding among alleles. We speculate that the disparity in structural constraints and ligand binding among the alleles within heterozygous individuals may contribute as a mechanism to the plumage color variation in the Coereba flaveola.

RESUMEN El melanismo en el color del plumaje se asocia frecuentemente al polimorfismo del receptor melanocortina-1. La ausencia de una estructura cristalográfica de la asociación entre la sustitución del Glutamato por Lisina en la posición 92 de la proteína MC1R y el plumaje completamente negro, no ha permitido tener un mejor entendimiento del efecto de mutaciones no sinónimas en la conformación y en el comportamiento en la membrana del MC1R. Examinamos la estructura asociada a la activación del receptor usando secuencias de ADN obtenidas del GenBank, para un modelamiento in silico de formas homólogas de la proteína. El modelo tridimensional muestra que la sustitución de Alanina por la Treonina en la posición 179 es un complemento estructural al efecto de reversión de carga asociado a la sustitución del Glutamato por Lisina en la posición 92 del MC1R. Proponemos la posibilidad de evolución gradual de la estabilidad y de propiedades electrostáticas del MC1R por la acumulación de estas substituciones. Estas perturban las propiedades fisicoquímicas que podrían ser necesarias para el plegamiento de las formas constitutivamente activas del MC1R sin alterar la afinidad de empalme con el ligando. La modelación computacional de la dinámica molecular de la afinidad de empalme del MC1R a la hormona estimulante de meloncitos predice la disparidad de la unión con el ligando entre alelos. Consideramos que posiblemente la disparidad entre alelos en heterocigotos en cuanto a restricciones estructurales y la unión con el ligando podría contribuir a la variación en el color del plumaje en Coereba flaveola.

Short-term outcomes of mitral valvuloplasty for Barlow disease in children / 中国胸心血管外科临床杂志

@#Objective    To explore the clinical effect of mitral valvuloplasty on children with Barlow disease combined with moderate to severe or severe mitral regurgitation. Methods    The clinical data of 10 patients with Barlow disease combined with moderate to severe or severe mitral regurgitation in Fuwai Hospital from January 2014 to August 2019 were analyzed retrospectively, including 3 males and 7 females, with a mean age of 8.7±7.9 years. Echocardiography before and during the operation confirmed that the mitral valve leaflets were long and swinging, the valve leaflets and the opposite edge protruded into the left atrium and were higher than the level of the mitral valve rings, the mitral valve rings were dilated, the papillary muscles and tendons were long, and the pathological changes after the operation showed mucoid degenertion of the valve leaflets and tendons, and some fibrous foci hyperplasia. Mitral valve repair included implantation of artificial valve ring, implantation of artificial tendon, posterior leaflets sliding, partial resection of posterior leaflets (excluding sliding), valve leaflets folding, tendon folding, papillary muscle splitting and annular valve contraction (excluding artificial valve ring implantation). The technique of mitral valve repair, early clinical results and follow-up echocardiographic data were analyzed. Results    All the patients successfully completed the mitral valve repair. The mean time of aortic occlusion was 73.2±17.4 min, and cardiopulmonary bypass time was 99.5±19.8 min. At the same time, 4 patients received tricuspid valve repair and 1 funnel chest correction. There was no reoperation in perioperative period. The 1-year and 5-year survival rates were 100.0% and 100.0%, respectively. The incidence of below moderate mitral regurgitation was 90.0% at postoperative 1 year and 72.0% at postoperative 5 years. Conclusion    For the young children who have Barlow disease and mitral regurgitation, considering the characteristics of heavy lesions, small operation space, and the need to meet the growth and development of valve, it is suggested to adopt the surgical techniques different from those of older children, such as valve ring retraction and tendon folding, if necessary, to adopt "edge to edge" suture, which can shorten aortic occlusion time and achieve good early effects, and its long-term effects still need further follow-up observation. Mitral valvuloplasty technique for Barlow disease similar to that of adults can be used in older children, including implantation of artificial valve ring and implantation of artificial tendon, etc.

Propeptide-mediated protein folding: mechanism and its impact on lipase / 生物工程学报

The formation of most proteins consists of two steps: the synthesis of precursor proteins and the synthesis of functional proteins. In these processes, propeptides play important roles in assisting protein folding or inhibiting its activity. As an important polypeptide chain coded by a gene sequence in lipase gene, propeptide usually functions as an intramolecular chaperone, assisting enzyme molecule folding. Meanwhile, some specific sites on propeptide such as glycosylated sites, have important effect on the activity, stability in extreme environment, methanol resistance and the substrate specificity of the lipase. Studying the mechanism of propeptide-mediated protein folding, as well as the influence of propeptide on lipases, will allow to regulate lipase by alternating the propeptide folding behavior and in turn pave new ways for protein engineering research.

Clinical features and tubulin folding cofactor E gene analysis in Iranian patients with Sanjad-Sakati syndrome / Características clínicas e análise genética e de cofatores de dobramento da tubulinaem pacientes iranianos com síndrome de Sanjad-Sakati

Abstract Objective Permanent hypoparathyroidism can be presented as part of genetic disorders such as Sanjad-Sakati syndrome (also known as hypoparathyroidism—intellectual disability-dysmorphism), which is a rare autosomal recessive disorder. Our aim was to confirm the diagnosis of a group of patients with dysmorphism, poor growth, and hypoparathyroidism clinically labeled as Sanjad-Sakati syndrome and to identify for the first time the genetic variations on Iranian patients with the same ethnic origin. Methods In this study, 29 cases from 23 unrelated Arab kindreds with permanent hypoparathyroidism and dysmorphism indicating Sanjad-Sakati syndrome were enrolled for 10 years in the southwest of Iran. The mutational analysis by direct sequencing of the tubulin folding cofactor E gene was performed for the patients and their families, as well as their fetuses using genomic DNA. Results Twenty-eight out of 29 cases had parental consanguinity. Twenty-seven cases presented with hypocalcemia seizure and two were referred because of poor weight gain and were found to have asymptomatic hypocalcemia. The dysmorphic features, hypocalcemia in the setting of low to normal parathyroid hormone levels and high phosphorus led to the diagnosis of these cases. Sequencing analysis of the tubulin folding cofactor E gene revealed a homozygous 12-bp deletion (c.155-166del) for all patients. Following that, prenatal diagnosis was performed for eight families, and two fetuses with a homozygous 12-bp deletion were identified. Conclusion These results make it much easier and faster to diagnose this syndrome from other similar dysmorphisms and also help to detect carriers, as well as prenatal diagnosis of Sanjad-Sakati syndrome in high-risk families in this population.

Resumo Objetivo O hipoparatireoidismo permanente pode estar presente como parte das doenças genéticas como na síndrome de Sanjad-Sakati (também chamada de síndrome de hipoparatireoidismo, retardo e dismorfismo), que é um distúrbio autossômico recessivo raro. Nosso objetivo foi confirmar o diagnóstico de um grupo de pacientes com dismorfismo, crescimento deficiente e hipoparatireoidismo clinicamente identificado como síndrome de Sanjad-Sakati e identificar as variações genéticas, pela primeira vez, em pacientes iranianos com a mesma origem étnica. Métodos Neste estudo, foram inscritos 29 casos de 23 famílias árabes sem parentesco com hipoparatireoidismo e dismorfismo indicando síndrome de Sanjad-Sakati, durante 10 anos no sudoeste do Irã. Foi feita a análise mutacional por sequenciamento direto do gene do cofator E de dobramento da tubulina dos pacientes e de suas famílias e também de seus fetos com o DNA genômico. Resultados Apresentaram consanguinidade parental 28 dos 29 casos. Desses, 27 casos apresentaram convulsão por hipocalcemia e dois foram encaminhados devido ao baixo ganho de peso, considerando diagnóstico de hipocalcemia assintomática. As características dismórficas, hipocalcemia na configuração de níveis de hormônio da paratireoide baixos a normais e alto nível de fósforo levaram ao diagnóstico dos casos. A análise de sequenciamento do gene do cofator E de dobramento da tubulina revelou deleção homozigótica de 12 pares de base (pb) (c.155-166del) em todos os pacientes. Após isso, foi feito o diagnóstico pré-natal em oito famílias e dois fetos foram identificados com deleção homozigótica de 12 pb. Conclusão Esses resultados tornam o diagnóstico dessa síndrome muito mais fácil e rápido do que outros dismorfismos semelhantes e também ajudam a detectar portadores, bem como o diagnóstico pré-natal da síndrome de Sanjad-Sakati em famílias de alto risco nessa população.

Cortical Folding On Medial Surface Of Foetal Brain At Different Gestational Age

Background: Cortical folding is a hallmark of many but not all mammalian brains. The degree of folding graduallyincreases with the size of brains in mammals but at different range between the families.Gyrification is a processwhich varies widely in mammals in early foetal and prenatal life.Materials: This study was conducted on 100 dead foetuses in anatomy department, brought from the departmentof Obstetrics and gynaecology of MNR Medical College and Hospital.Results: The brain surface is smooth up to 12 weeks,Cingulate sulcus appeared by 16-18weeks.Growth of adjoininglobes of brain make surface more convoluted with well-defined sulci and gyral pattern between 30-32 weeks.Conclusion: Cortical folding is due to consequence of restricted space and rapid growth of brain with in thecranial cavity. There is no differences between male and female brains of same gestational age, with no obviousasymmetrical development of gyri on different lobes of brain

Enhance the affinity of nanobodies to CD47 antigen by fusion expression of COMP48 self-folding peptide / 国际生物医学工程杂志

Objective To modify CD47 nanobody with the self-folding peptide human cartilage oligomeric matrix protein (COMP48) so as to enhance its affinity to CD47 antigen. Methods The fusion sequences of COMP48 and CD47 nanobody (VHHB1) were designed and synthesized, and the recombinant plasmid pET22b-VHHB1-COMP48 was constructed and transformed into E. coli BL21 (DE3) to induce expression of the fusion protein. The binding specificity and affinity of the fusion protein and the antigen CD47 were detected by Western Blot, indirect enzyme-linked immunosorbent assay (ELISA) and non-competitive ELISA. Results The recombinant VHHB1-COMP48 was expressed in BL21(DE3) by inducing with 1 mmol/L IPTG and purified at 90％homogenous in IMAC. Western Blot results showed that the recombinant protein VHHB1-COMP48 specifically binds to antigen CD47 but not to unrelated protein. The indirect ELISA and non-competitive ELISA results showed that the affinity of the conjugated recombinant protein VHHB1-COMP48 was enhanced compared to that of the non-conjugated nanobody, and the difference was statistically significant ( P<0 . 01 ) . Through non-competitive ELISA , the constants of affinity and dissociation constants were 6.97 ×107 L/mol and 1.434 ×10-8 mol/L, respectively. Conclusions The affinity of the nanobody for the antigen can be improved by conjugating a human cartilage matrix protein (COMP48) after the nanobody.

Immediate Nail Lengthening with the Eponychial Folding Procedure in Acute Finger Tip Injury

PURPOSE: We present the clinical results and operative method of the immediate eponychium of nail fold set back for lengthening of nails caused by acute fingertip injuries. METHODS: The research was conducted with a total of 172 patients during the period from January 2014 to June 2016. The operation method was performed in a way to fold down the two sides of the nail eponychium and had suture. A survey of the patients' subjective satisfaction was conducted and the relative nail length was compared before and after the operation as well as the nail length of the uninjured contralateral finger. The mean follow-up period was 18.2 weeks. RESULTS: In all cases, the operation time was under 3 minutes. There were no specific complications such as nail eponychium's necrosis or congestion. The new nail did not have any additional deformation. On average, the extended nail length was 3.2 mm. Compared with preoperation, the average extension ratio of the nail length was 48%, even with 75% of nail length recovery in comparison with the uninjured contralateral finger. The subjective self-satisfaction score was 92.5 on average. The satisfaction score was higher for patients who had greater remnant nail length. CONCLUSION: Immediate nail lengthening with the eponychial folding is a simple, safe and useful method with high subjective satisfaction in aesthetics for the patients with acute fingertip injuries.

Strategies to improve the folding and modification of recombinant proteins: a review / 生物工程学报

Gene expression technology has made great progress with the continuous developments and researches of molecular biology. Though many systems to produce recombinant proteins have been studied, none of them is available so far to satisfy the needs completely. With the increasing demands of bioactive peptides and protein drugs, expression quantity and correct posttranslational folding and modifications are also needed under the circumstance which can make proteins more close to native conformation and higher activity and stability. Based on our previous work, we summarized the factors affecting the folding and modifications of recombinant proteins correctly from five aspects, including expression system and hosts, secretory expression, coexpression, fusion expression, and the culture conditions, as well as improvement strategies.

Synthesis of soluble proinsulin in a cell-free protein synthesis system / 生物工程学报

Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) μg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.

Efficacy comparison of combined and single operation for senile lower eyelid entropion / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To compare the clinical effect of the lower eyelid retractor muscle transposition and eyelid orbicularis muscle folding shorten combined with lower eyelid retractor muscle transposition in the treatment of senile lower eyelid entropion. <p>METHODS: Sixty-four cases(85 eyes)with senile lower entropion were divided into Group A(31 cases 42 eyes)and Group B(33 cases 43 eyes)according to the different ways of operation from January 2013 to October 2014 in our hospital. Patients in Group A were treated by eyelid retractor muscle transposition while patients in Group B treated by eyelid orbicularis muscle folding shortening combined with lower eyelid retractor muscle transposition. The short-term and long-term therapeutic efficacy and two-year recurrence rates after operation were compared between the two groups. <p>RESULTS: The short-term effective rate of patients in Group B was higher than that of Group A while the difference was not statistically significant(98% <i>vs</i> 95%, <i>P</i>>0.05). The long-term effective rate of patients in Group B was higher than that of Group A and the difference was statistically significant(95% <i>vs </i>83%)and two-year recurrence rate of patients in Group B was lower than that of Group A(5% <i>vs</i> 17%)and the difference was statistically significant(<i>P</i><0.05). <p>CONCLUSION:The clinical curative effect of eyelid orbicularis muscle folding shortening combined with lower eyelid retractor muscle transposition is better than single retractor muscle transposition in the treatment of senile lower eyelid entropion.

Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain

β/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various β/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in βB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of βB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native βB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of βB1-crystallin on βA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native βB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all β/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of β/γ-crystallin domains by protecting the hydrophobic core from solvent access.

Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis

Background The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.(AU)

Processamento intracelular da fibrilina-1 mutada na síndrome de Marfan: escape do controle de qualidade pela dissulfeto isomerase proteica / Mutated fibrillin-1 intracellular processing in Marfan syndrome: bypass of a protein disulfide isomerase-mediated quality control

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT...

Marfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT...

Comparing Clinical Outcomes of Descemet's Membrane Stripping Automated Endothelial Keratoplasty Between Graft Insertion Methods

PURPOSE: To compare clinical outcomes of Descemet's stripping automated endothelial keratoplasty (DSAEK) between different graft insertion methods. METHODS: The clinical records of 32 eyes of 30 DSAEK patients were retrospectively analyzed. Patients were divided into 2 groups according to graft insertion method. Group A: Taco-folding, group B: Tan-endoglide. The best corrected visual acuities (BCVA), intraocular pressures, astigmatism, endothelial cell count, central corneal thickness and complications were evaluated pre and post-operatively. RESULTS: The average follow-up period was 19 months (range 1-67). Postoperative log MAR visual acuity had significantly improved both from 1.63 (log MAR) to 0.69 and 0.53 at 12 months in each group (p = 0.035, p = 0.000). Mean endothelial cell survival of each group at 1 month postoperative were 75.8% (range 62.7-88.6) and 87.7% (range 70.2-97.9), respectively (p = 0.012). The differences of BCVA improvement and endothelial cell survival between the groups at 12 months were not significant (p = 0.393, p = 0.544). CONCLUSIONS: Both methods showed fast visual recovery. Using Tan-endoglide insertion resulted less endothelial cell loss at early post-operative period and showed less post-operative complication and graft failure.