Regional structural and viscoelastic properties of fibrocartilage upon dynamic nanoindentation of the articular condyle

Fibrocartilage,a tissue with macromaterial properties between dense fibrous tissue and hyaline cartilage, is not well understood in its ultrastructure and regional viscoelastic properties. Here nanoindentation with atomic force microscopy was performed on fresh fibrocartilage samples of rabbit jaw joint condyles. Each sample was divided into anteromedial, anterolateral, posteromedial, and posterolateral regions for probing and topographic imaging in 2 x 2 microm and 10 x 10 microm scan sizes. Young's moduli differed significantly among these regions in a descending gradient from the anteromedial (2.34 +/- 0.26 MPa) to the posterolateral (0.95 +/- 0.06 MPa). The Poisson ratio, defined as lateral strain over axial strain, had the same gradient distribution: highest for the anteromedial region (0.46 +/- 0.05) and lowest for the posterolateral region (0.31 +/- 0.05). The same four regions showed a descending gradient of surface roughness: highest for the anteromedial (321.6 +/- 13.8 nm) and lowest for the posterolateral (155.6 +/- 12.6 nm). Thus, the regional ultrastructural and viscoelastic properties of fibrocartilage appear to be coregulated. Based on these region-specific gradient distributions, fibrocartilage is constructed to withstand tissue-borne shear stresses, which likely propagate across its different regions. A model of shear gradient and concentric gradient is proposed to describe the region-specific capacity of fibrocartilage to sustain shear stresses in tendons, ligaments, joints, and the healing bone across species.     

Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket

Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50-residue region near the C-terminus of intimin. Alanine-scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin-Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin-Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.     

Two fatty acids can replace one phospholipid in condensed complexes with cholesterol

We report that the monolayer phase diagram for binary mixtures of dimyristoylphosphatidylethanolamine (DMPE) and dihydrocholesterol (DChol) is largely unchanged when each phospholipid molecule is replaced by two myristic acid (MA) molecules or various mixtures of the lysophospholipid and myristic acid. The corresponding phase diagrams all show the formation of "condensed complexes" of DChol and lipid. The condensed complex stoichiometry is thus largely determined by the C14 fatty acid acyl chains, in this case about 4-4.6 per DChol molecule.     

Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation

Monoubiquitylation is a well-characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin-binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded alpha-helices that bind ubiquitin independently, non-cooperatively and with modest affinity. The Vps27 N-terminal UIM engages the Leu8-Ile44-Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8-Ile44-Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin-binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.     

Testicular fine needle aspiration cytology for the diagnosis of azoospermia and oligospermia

OBJECTIVE: To evaluate qualitative and quantitative cytologic features on testicular fine needle aspiration biopsy in the diagnosis of azoospermia and oligospermia and to correlate cytologic and histologic diagnoses. STUDY DESIGN: In this prospective study, 50 infertile males selected from the infertility clinic of Guru Tegh Bahadur Hospital were studied. Fine needle aspiration cytology (FNAC) smears from both testes of 27 azoospermic and 23 oligospermic patients (sperm count < 10 million per milliliter) were stained with May-Grünwald-Giemsa and Papanicolaou stain. Differential counting of 500 spermatogenic cells was done, and the number of Sertoli cells per 500 germ cells was determined for calculating the spermatic index and Sertoli cell index, respectively. FNAC and testicular biopsy were performed under local anesthesia as a minor surgical procedure. RESULTS: Six groups were identified on FNAC smears from azoospermic patients: I. normal spermatogenesis (8), II. hypospermatogenesis (2), III. maturation arrest (2), IV. Sertoli cells only (6), V. atrophic pattern (7), and VI. Leydig cell predominance (2). In oligospermic patients two groups were identified: I. those with normal spermatogenesis (4), and II. those with subnormal spermatogenesis (19). Correlation with histopathologic examination was seen in 81.5% azoospermic and 65.2% oligospermic patients. CONCLUSION: Qualitative and quantitative evaluation of testicular FNAC provides useful information on both azoospermic and oligospermic patients. FNAC performed under local anesthesia is an acceptable outpatient procedure that consistently yields sufficient diagnostic material in all patients.     

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone¹ H^N, ¹⁵N,¹³ C and¹³ C and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.     

Energetic salts from nitroformate ion

Development of new energetic salts is the key factor in replacing low performance compounds in conventional formulations of high explosives as well as propellants. Ten salts based on the nitroformate anion and various nitrogen-rich cations were designed and their geometric optimizations carried out using the density functional method. With reasonable oxygen balance (from ?36 % to 0 %), heats of formation (47–624 kJ mol^?1) and high densities (1.81–1.89 g cm^?3), the detonation velocity (D) and pressure (P) values of salts were calculated as 8.62–9.36 km s^?1 and 33.10–40.01 GPa, respectively. Lastly, the nitroformate salts studied in this work are of prospective interest as high performance explosives.

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Graphical Abstract Formation of nitroformate salt from nitroformate anion and a nitrogen-rich cation

     

Vasopressin Infusion with Small-Volume Fluid Resuscitation during Hemorrhagic Shock Promotes Hemodynamic Stability and Survival in Swine

Introduction

Current management of hemorrhagic shock (HS) in the battlefield and civilian settings favors small-volume fluid resuscitation before controlling the source of bleeding. We investigated in a swine model of HS the effects of vasopressin infusion along with small-volume fluid resuscitation; with erythropoietin (EPO) and HS severity as additional factors.

Methods

HS was induced in 24 male domestic pigs (36 to 41 kg) by blood withdrawal (BW) through a right atrial cannula modeling spontaneous bleeding by a mono-exponential decay function. The initial 12 pigs received no fluids; the last 12 pigs received normal saline (NS) half the BW volume. Pigs were randomized 2:1 to receive intraosseously vasopressin (0.04 U/kg·min^-1) or vehicle control from minute 7 to minute 210. Pigs assigned to vasopressin were further randomized 1:1 to receive EPO (1,200 U/kg) or vehicle control and 1:1 to have 65% or 75% BW of their blood volume. Shed blood was reinfused at 210 minutes and the pigs recovered from anesthesia.

Results

Survival at 72 hours was influenced by vasopressin and NS but not by EPO or % BW. Vasopressin with NS promoted the highest survival (8/8) followed by vasopressin without NS (3/8), NS without vasopressin (1/4), and neither treatment (0/4) with overall statistical significance (log-rank test, p = 0.009) and each subset different from vasopressin with NS by Holm-Sidak test. Vasopressin increased systemic vascular resistance whereas NS increased cardiac output.

Conclusion

Vasopressin infusion with small-volume fluid resuscitation during severe HS was highly effective enabling critical hemodynamic stabilization and improved 72 hour survival.