Cloning and Expression of Dynorphin-32 in E. coli and Analgesic Tests in Mice Models

Chronic severe pain is most destructive to the human psyche. Presently available analgesics are effective in the treatment of acute pain, although even in this setting “analgesic gaps” remain. But for chronic pain, there is a need to develop better therapeutic strategies and analgesics than are currently available. Epidemiologic data indicates that even in prosperous, developed nations, 40 % of patients with chronic pain are only partially satisfied and 15 % are not satisfied at all with the treatments available to them. At the same time, proper treatment of pain is increasingly in demand as a human right by patients, their families, and governments. Therefore, modern medicine urgently needs more effective treatments for pain. Therefore, the challenge of this decade is to produce small molecules which mimic chemical drugs, in order to overcome the ineffectiveness of currently available analgesic drugs. This work includes identification of target receptors, selection of potent existent drugs, modelling, and selection of peptide based therapeutic drugs.METHODSThe selected mutant sequences of analgesic peptides were codon optimized using DNA 2.0 software for maximum expression in Saccharomyces cerevisiae and E. coli. Codon optimization was carried out at above 15% threshold and as per the codon usage frequency of standard Saccharomyces and E. coli tables. For yeast Alpha F mating tag and for E. coli ompA tag was used for efficient extracellular recombinant expressions. The expression studies were carried out initially in E. coli with analgesic peptides. Lac promoters were used for induction of the genes of these analgesic peptides. These peptides were also precipitated using acetone and used for the analysis. Partially purified analgesic peptides by salt and acetone precipitation were used for bioassays by Tail immersion and hot plat assays.RESULTSPositive confirmed constructs were transformed in BL21 for expression. Dynorphin recombinants in BL21 were used for expression studies. The expression of recombinant protein was confirmed by 16% SDS PAGE. The over expressed peptide was first precipitated by ammonium sulphate and dialyzed. The dialyzed samples were used for bioassay studies. Fentanyl was used as positive control and uninduced culture broth as negative control. Among the three peptides studied JV5 (Peptide F) showed 62% central analgesic activity (observed by tail immersion assay) when compared to fentanyl by oral route of administration and fentanyl treatment (hot plate assay) indicating similar action suggesting usage of this peptide as strong analgesic molecule for future pain treatments.

Leukotriene B4 in equine asthma syndrome: what do we know so far? / Leucotrieno B4 na síndrome asmática equina: o que sabemos até o momento?

The term "equine asthma syndrome" (EAS) was recently proposed due to the resemblance of the equine disease to human asthma. Leukotrienes cause constriction of the bronchi, especially in the lower airways and increase mucus secretion in the respiratory system. Leukotriene B4 (LTB4) has been discovered as a strong chemotactic factor, which plays a role in neutrophil migration. The immunologic background of EAS remains not fully elucidated despite many studies on the pathogenesis. This study aimed to evaluate the LTB4 concentration in the bronchoalveolar lavage fluid (BALF) of horses with and without pulmonary inflammatory disease. Thirty-five mixed breed horses were studied and LTB4 was determined by using specific ELISA Kit. The horses were grouped by 2 different criteria for statistical analysis of data: 1) according to the values for BALF citology and 2) according to the detection of LTB4 in BALF. There was significant difference of effect of age on the LTB4 detection in equine BALF. Younger animals were the majority where it was possible to detect LTB4 values in LBA. In conclusion, there was an effect of age on the detection of LTB4 in equine BALF, where LTB4 levels were more easily detected in younger animals than older animals and the results of this study raise the possibility of considering future studies with the objective of establishing the real role and the best moment to detect LTB4 in BALF of the equine asthma syndrome.(AU)

Recentemente, o termo "síndrome da asma equina" (SAE) foi proposto devido à semelhança da doença equina à asma humana. Os leucotrienos causam constrição dos brônquios, especialmente nas vias aéreas posteriores e aumentam a secreção de muco no sistema respiratório. O leucotrieno B4 (LTB4) foi descoberto como um forte fator quimiotático, que desempenha um papel na migração de neutrófilos. O fundo imunológico do SAE permanece não completamente elucidado apesar de muitos estudos sobre a patogênese. Este estudo teve como objetivo avaliar a concentração de LTB4 no lavado broncoalveolar (LBA) de equinos com e sem doença inflamatória pulmonar. Trinta e cinco cavalos de raças mistas foram estudados e o LTB4 foi determinado usando o kit ELISA específico. Os animais foram agrupados por dois critérios diferentes para análise estatística dos dados: 1) de acordo com os valores para citologia do LBA e 2) de acordo com a detecção do LTB4 no LBA. Houve diferença significativa do efeito da idade na detecção do LTB4 no LBA equino. Os animais mais jovens foram a maioria onde foi possível detectar os valores de LTB4 no LBA. Em conclusão, houve um efeito da idade na detecção de LTB4 em LBA equino, onde os níveis de LTB4 foram mais facilmente detectados em animais jovens do que em animais mais velhos e foi possível detectar a concentração de LTB4 no LBA equino e os resultados deste estudo levantam a possibilidade de considerar futuros estudos com o objetivo de estabelecer o real papel e o melhor momento para detectar LTB4 no LBA da síndrome asmática equina.(AU)

Experience of RAO Xiang-rong in Treating Early Diabetic Kidney Diseases / 中国中医药信息杂志

This article analyzed the clinical experience of Professor RAO Xiang-rong in the treatment of early diabetic kidney diseases (DKD) from the aspects of etiology, pathogenesis and methods of treatment prescriptions. Professor RAO believes that early DKD patients suffer from long-term eating disorders and like to eat high-salt, high-protein, high-fat, and high-calorie food, which results in the occlusion of middle Jiao, unruly dryness fire and retention of damp-heat, and then leads to the damage to spleen and kidney, qi and yin deficiency and blood stasis in meridians. He points out that the dryness-heat of middle Jiao is the core pathogenesis and puts forward the treatment concept of "Treating renal diabetes must first treat gastric diabetes".

Hip Surgery and Hip Joint Function : A Discussion with Anatomical Basis / The Japanese Journal of Rehabilitation Medicine

In Japan, hip joint degenerative diseases are based on acetabular dysplasia and the treatment strategy has been focused primarily on how to maintain the hip joint function and how to achieve pain relief. We employed total hip arthroplasty (THA) to treat end-stage OA of the hip joint. There are many possible complications in THA including infection, dislocation and nerve palsy. Especially in patients with Crowe IV dislocation, THA should be accompanied by simultaneous subtrochanteric shortening osteotomy to prevent sciatic nerve palsy. There is concern that the small metal head and posterior approach without re-attachment of posterior soft tissue commonly used in THA may be susceptible to postoperative dislocation. To avoid this scenario, we developed a modified less invasive total hip arthroplasty surgical approach that uses the anterolateral approach of the modified Watson-Jones approach. By using this approach, the risk of postoperative dislocation can be greatly reduced due to the intact posterior stabilizing soft tissue of the hip joint. Recently, a new concept in hip pathology describing femoroacetabular impingement (FAI) including retroversion of the acetabulum was proposed by Professor Ganz in 2003. Patients with acetabular dysplasia were also assumed to have acetabular retroversion. In patients with acetabular dysplasia to prevent further pathology of the hip joint, we developed a reorientation rotational acetabular osteotomy using a navigation system to obtain accurate direction and alignment in the reorientated hip joint. Orthopedic surgeons and rehabilitation doctors need to understand each other well in their respective fields of pathology and treatment strategy to ensure the optimal treatment of motor diseases.