Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR, and SPINK1 variants were associated with pancreatitis risk. We now report two significant genome-wide associations identified and replicated at PRSS1-PRSS2 (1×10-12) and x-linked CLDN2 (p < 1×10-21) through a two-stage genome-wide study (Stage 1, 676 cases and 4507 controls; Stage 2, 910 cases and 4170 controls). The PRSS1 variant affects susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous male) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men – male hemizygous frequency is 0.26, female homozygote is 0.07

Anisotropy of the passive and active rat vagina under biaxial loading

Pelvic organ prolapse, the descent of the pelvic organs from their normal anatomical position, is a common condition among women that is associated with mechanical alterations of the vaginal wall. In order to characterize the complex mechanical behavior of the vagina, we performed planar biaxial tests of vaginal specimens in both the passive (relaxed) and active (contracted) states. Specimens were isolated from virgin, female Long-Evans rats (n = 16) and simultaneously stretched along the longitudinal direction (LD) and circumferential direction (CD) of the vagina. Tissue contraction was induced by electric field stimulation (EFS) at incrementally increasing values of stretch and, subsequently, by KCl. On average, the vagina was stiffer in the CD than in the LD (p < 0.001). The mean maximum EFS-induced active stress was significantly higher in the CD than in the LD (p < 0.01). On the contrary, the mean KCl-induced active stress was lower in the CD than in the LD (p < 0.01). When comparing the mean maximum EFS-induced active stress to the mean KCl-induced active stress, no differences were found in the CD (p = 0.366) but, in the LD, the mean active stress was much higher in response to the KCl stimulation (p < 0.001). Collectively, these results suggest that the anisotropic behavior of the vaginal tissue is determined not only by collagen and smooth muscle fiber organization but also by the innervation

Ex-vivo measurement of the biaxial properties of rat vagina: some preliminary results

Pelvic floor disorders (PFDs) concern a wide range of disorders that occur when one of the organs of the reproductive system loses its functionality. To date, PFDs affect nearly 30 million women in the U.S. and the symptoms usually associated with these disorders include urinary and fecal incontinence, pain, psychological and emotional distress. While the exact etiology of PFDs is unknown, mechanical alterations to pelvic organs, supportive ligaments, fasciae, and muscles due to pregnancy, age, and weight gain contribute to the development of these disorders. There is an unmet clinical need to characterize the mechanical properties of the female reproductive organs and supporting tissues. One PFD is related to the decrease in the mechanical integrity of the vagina, which can cause the pelvic organs to descend into the vagina, a condition known as pelvic organ prolapse. Within this context, the aim of this work was to devise a biaxial testing system for assessing passive properties of rat vagina. While many studies characterized the uniaxial mechanical properties of vaginal tissues, biaxial mechanical properties are almost unexplored. Nonetheless, the nonlinear and anisotropic nature of vaginal tissue has already been shown in a few preliminary studies. In this study, the development of a testing system is presented. The system is tested using rubber specimens since the mechanical properties of rubber are well known. Finally, some preliminary data were collected using specimens from rat vagina. The results showed that the “toe region” of the tissue computed along the longitudinal axis is more extended than the counterpart measured along the circumferential axis, confirming the capability of the proposed system to properly measure the mechanical properties of vagina along the two main axes simultaneously

Anterior Cervical Discectomy With Fusion Using a Local Source for Cancellous Autograft: A Biomechanical Analysis of Vertebral Body Stability in an Osteopenic Bone Model

Background: Anterior cervical discectomy with fusion is an effective treatment for patients having cervical radiculopathy and myelopathy. To reduce morbidity associated with autograft taken from the iliac crest without sacrificing high fusion rates, a novel technique that harvests bone from the vertebral body adjacent to the operative disc space has been proposed. The effects of square and round bone graft harvest techniques on the mechanical stability of the osteopenic donor vertebrae are unknown. We analyzed the biomechanical implications of the technique by subjecting osteopenic models to uniaxial compression to compare yield strengths of surgically altered and unaltered specimens. Methods: Biomechanical grade polyurethane foam was cut into 60 different 12 mm × 17 mm × 20 mm blocks. The foam had a density of 10 pounds per cubic foot, simulating osteoporotic bone. Rectangular prism (4 mm × 4 mm × 6 mm) and cylindrical cores ( r = 2 mm, h = 8 mm) were removed from 20 blocks per group. Twenty samples were left intact as a control group. Anterior plate screws were applied to the models and a Polyether ether ketone (PEEK) interbody spacer was placed on top. Samples underwent uniaxial compression at 0.1 mm/s until mechanical failure. Points of structural failure were determined using a 0.1% offset on a force–displacement curve and compared to determine the reductions in compressive strength. Results: The mean force eliciting structural failure for intact samples was 450.6 N. Average failure forces for rectangular prisms and cylindrical cores removed were 383.2 and 395.4 N, respectively. Removal of a rectangular prismatic core of the necessary volume resulted in a 15.0% reduction in compressive strength, while removal of a cylindrical core of comparable volume facilitated a reduction of 12.2%. Conclusion: Local autograft harvested from adjacent vertebrae reduces morbidity associated with a second surgical site while minimally reducing the compressive strength of the donor vertebra in an osteopenic model, lending credence to the efficacy of this technique in elderly patient populations

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rarePRSS1,CFTR, andSPINK1variants were associated with pancreatitis risk. We now report two significant genome-wide associations identified and replicated atPRSS1-PRSS2(1×10-12) and x-linkedCLDN2(p < 1×10-21) through a two-stage genome-wide study (Stage 1, 676 cases and 4507 controls; Stage 2, 910 cases and 4170 controls). ThePRSS1variant affects susceptibility by altering expression of the primary trypsinogen gene. TheCLDN2risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous male)CLDN2genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men – male hemizygous frequency is 0.26, female homozygote is 0.07