Rolling adhesion kinematics of yeast engineered to express selectins

Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.     

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.     

Preferential proliferation and differentiation of double-positive thymocytes into CD8(+) single-positive thymocytes in a novel cell culture medium

The identification of factors that regulate the proliferation and differentiation of double-positive (DP) into CD4(+) and CD8(+) single-positive (SP) thymocytes has proven difficult due to the inability of DP thymocytes to proliferate, expand, and differentiate into SP thymocytes in available cell culture media. Here we report on the ability of DP thymocytes to differentiate in a novel conditioned medium, termed XLCM, derived from the supernatant of mitogen activated human cord blood mononuclear cells. During a 5-day culture in XLCM in the absence of thymic stromal cells, DP thymocytes from normal mice and MHC double knockout mice (lack SP thymocytes) proliferate, expand, and differentiate into several (alphabetaTCR(+), NK1.1(+)alphabetaTCR(+), and gammadeltaTCR(+)) subsets of CD4(+) and predominantly CD8(+) SP thymocytes. These studies suggest that the use of XLCM may aid in the characterization of factors that regulate the differentiation of DP thymocytes into CD8(+) SP thymocytes.     

Auxin — Calcium Interaction in Adventitious Root Formation in the Hypocotyl Explants of Sunflower (Helianthus annuus L.)

Auxin-calcium interaction has been studied to understand their involvement in adventitious root initiation from the hypocotyl explants of sunflower (Helianthus annuus L.). When hypocotyl explants were cultured on MS medium (containing calcium), 1 mg l^-1 IAA was found to be optimal for root induction. However, the hypocotyl explants washed in EGTA (10_-5M) solution for the removal of extracellular calcium, when cultured on medium containing IAA and calcium, exhibited enhanced rooting response. When EGTA-washed explants were cultured on the medium supplemented with lanthanum chloride (10^-6 and 10^-5M), it resulted in the inhibition of the rooting response and this inhibitory effect could be alleviated by the simultaneous addition of IAA. Similar observations have been made by using calcium channel blockers, verapamil and TMB-8, and also a calmodulin inhibitor, trifluoperazine. A net influx of extracellular calcium in the differentiating cells is thus presumed to accompany the auxin-induced response. These results have been discussed in light of initial lack of polarity in the decapitated hypocotyl segments subjected to auxin treatment.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

SBOL Visual: A Graphical Language for Genetic Designs

Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.