Quantitative phase retrieval with low photon counts using an energy resolving quantum detector.

X-ray phase contrast imaging (PCI) combined with phase retrieval has the potential to improve soft-material visibility and discrimination. This work examined the accuracy, image quality gains, and robustness of a spectral phase retrieval method proposed by our group. Spectroscopic PCI measurements of a physical phantom were obtained using state-of-the-art photon-counting detectors in combination with a polychromatic x-ray source. The phantom consisted of four poorly attenuating materials. Excellent accuracy was demonstrated in simultaneously retrieving the complete refractive properties (photoelectric absorption, attenuation, and phase) of these materials. Approximately 10 times higher SNR was achieved in retrieved images compared to the original PCI intensity image. These gains are also shown to be robust against increasing quantum noise, even for acquisition times as low as 1 s with a low-flux microfocus x-ray tube (average counts of 250 photons/pixels). We expect that this spectral phase retrieval method, adaptable to several PCI geometries, will allow significant dose reduction and improved material discrimination in clinical and industrial x-ray imaging applications.

Auditing the Representation of Female Athletes in Sports Medicine Research: Achilles Repair.

Background: Establishing evidence-based recommendations specific to female athletes has been overlooked in sports medicine. Achilles tendon rupture is one of the most common musculoskeletal injuries, occurring in 15 to 55 per 100 000 people annually. Differences in injury rates could be due to hormonal effects, as estrogen receptors have been identified in tendons along with decreased tendon strain based on oral contraceptive use. The primary purpose of this study was to audit the representation of female athletes in the literature regarding Achilles repair. Methods: An electronic search was performed using PubMed to identify articles related to Achilles repair using the protocol by Smith et al. Studies were assessed by population, size, athletic caliber, study impact, research theme, and menstrual status. Results: Female representation across all studies was 1783 of 10 673 subjects (16.7%). Composition of included studies was predominantly mixed-sex cohorts with 131 of 169 (77.5%) included studies. Within mixed-sex cohort studies, the total representation of female athletes was 1654 of 8792 participants (18.9%). Thirty-two studies were male only, constituting 1540 participants, whereas 3 studies were female only composed of 86 athletes. Importantly, the disparity between male and female representation worsened as the athletic caliber of the study population increased, with 5.0% female representation in studies with professional athletes. No study collected data related to menstrual status and its potential relationship to Achilles rupture or postoperative outcomes. Conclusion: Mixed-sex cohort studies underrepresented female athletes, and male-only cohort studies were more common than female-only studies. These findings indicate a need for increased representation of female athletes as well as acknowledgment of menstrual status in research related to Achilles repair. Future studies should focus on representation of female athletes and data collection related to sex-specific hormones, hormonal contraceptive use, and menstrual status to improve treatment of Achilles tendon ruptures for female athletes. Level of Evidence: Level IV, case series.

Traumatic Peroneal Nerve Injuries: A Systematic Review.

BACKGROUND: The common peroneal nerve (CPN) is the most commonly injured peripheral nerve of the lower extremity in patients with trauma. Traumatic CPN injuries have historically been associated with relatively poor outcomes and patient satisfaction, although improved surgical technique and novel procedures appear to improve outcomes. Given the variety of underlying injury modalities, treatment options, and prognostic variables, we sought to evaluate and summarize the current literature on traumatic CPN injuries and to provide recommendations from an analysis of the included studies for treatment and future research. METHODS: A systematic review was performed using PubMed, Embase, and Cochrane databases per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Search terms consisted of variations of "peroneal nerve" or "fibular nerve" combined with "injury," "laceration," "entrapment," "repair," or "neurolysis." Information with regard to treatment modality, outcomes, and patient demographic characteristics was recorded and analyzed. RESULTS: The initial search yielded 2,301 articles; 42 met eligibility criteria. Factors associated with better outcomes included a shorter preoperative interval, shorter graft length when an interposed graft was used, nerve continuity, and younger patient age. Gender or sex was not mentioned as a factor affecting outcomes in any study. Motor grades of ≥M3 on the British Medical Research Council (MRC) scale are typically considered successful outcomes. This was achieved in 81.4% of patients who underwent neurolysis, 78.8% of patients who underwent end-to-end suturing, 49.0% of patients who underwent nerve grafting, 62.9% of patients who underwent nerve transfer, 81.5% of patients who underwent isolated posterior tibial tendon transfer (PTTT), and 84.2% of patients who underwent a surgical procedure with concurrent PTTT. CONCLUSIONS: Studies included in this review were heterogenous, complicating our ability to perform further analysis. It is not possible to uniformly advocate for the best treatment option, given diverse injury modalities and patient presentations and a variety of prognostic factors. Many studies do not show outcomes with respect to injury modality. Future studies should show preoperative muscle strengths and should clearly define outcomes based on the injury modality and surgical treatment option. This would allow for greater analysis of the most appropriate treatment option for a given mechanism of injury. Newer surgical techniques are promising and should be further explored. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Evaluation of two point of care technologies for measuring monoclonal antibody therapeutic-A concentrations in blood.

Aim: Current blood monitoring methods require sample collection and testing at a central lab, which can take days. Point of care (POC) devices with quick turnaround time can provide an alternative with faster results, allowing for real-time data leading to better treatment decisions for patients. Results/Methodology: An assay to measure monoclonal antibody therapeutic-A was developed on two POC devices. Data generated using 75 serum samples (65 clinical & ten spiked samples) show correlative results to the data generated using Gyrolab technology. Conclusion: This case study uses a monoclonal antibody therapeutic-A concentration assay as an example to demonstrate the potential of POC technologies as a viable alternative to central lab testing with quick results allowing for real-time decision-making.

The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation.

Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5.

During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.

Immature hematopoietic cells display selective requirements for adhesion- and degranulation-promoting adaptor protein in development and homeostatsis.

Adhesion- and degranulation-promoting adaptor protein (ADAP) modulates T cell development and function and promotes TCR signaling. Regulation of ADAP protein expression during thymopoiesis and in development of other hematopoietic lineages has not been explored. Using intracellular staining, we detected ADAP protein in bone marrow lymphocyte precursors. Like its binding partner SH2-containing leukocyte phosphoprotein of 76 kDa, ADAP is dynamically regulated during thymocyte positive selection. ADAP is also found in unconventional thymocytes, including NKT, CD8alphaalpha, and TCRgammadelta T cells. In peripheral T cells, ADAP is up-regulated after TCR stimulation and with acquisition of memory status. Although absent in splenic B cells, ADAP is present in pro-B cells, as well as in BM erythrocyte and myeloid progenitors. Studies with radiation chimeras show that ADAP is dispensable for NKT, CD8alphaalpha and TCRgammadelta T cell development, while confirming that ADAP is required for optimal development of conventional TCRalphabeta T cells in the thymus. Interestingly, ADAP is necessary for CD8alphaalpha homeostasis in the small intestinal epithelium, yet is dispensable for optimal reconstitution of splenic B cell populations. Our observations highlight the dynamic regulation of ADAP during T cell maturation and document expression patterns that suggest a possible role for ADAP in development of non-T hematopoietic lineages.

Attenuation of IL-7 receptor signaling is not required for allelic exclusion.

Allelic exclusion prevents pre-B cells from generating more than one functional H chain, thereby ensuring the formation of a unique pre-BCR. The signaling processes underlying allelic exclusion are not clearly understood. IL-7R-dependent signals have been clearly shown to regulate the accessibility of the Ig H chain locus. More recent work has suggested that pre-BCR-dependent attenuation of IL-7R signaling returns the H chain loci to an inaccessible state; this process has been proposed to underlie allelic exclusion. Importantly, this model predicts that preventing pre-BCR-dependent down-regulation of IL-7R signaling should interfere with allelic exclusion. To test this hypothesis, we made use of transgenic mice that express a constitutively active form of STAT5b (STAT5b-CA). STAT5b-CA expression restores V(D)J recombination in IL-7R(-/-) B cells, demonstrating that IL-7 regulates H chain locus accessibility and V(D)J recombination via STAT5 activation. To examine the effects of constitutively active STAT5b on allelic exclusion, we crossed STAT5b-CA mice (which express the IgM(b) allotype) to IgM(a) allotype congenic mice. We found no difference in the percentage of IgM(a)/IgM(b)-coexpressing B cells in STAT5b-CA vs littermate control mice; identical results were observed when crossing STAT5b-CA mice with hen egg lysozyme (HEL) H chain transgenic mice. The HEL transgene enforces allelic exclusion, preventing rearrangement of endogenous H chain genes; importantly, rearrangement of endogenous H chain genes was suppressed to a similar degree in STAT5b-CA vs HEL mice. Thus, attenuation of IL-7R/STAT5 signaling is not required for allelic exclusion.

Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment.

The molecular mechanisms regulating lymphocyte lineage commitment remain poorly characterized. To explore the role of the IL7R in this process, we generated transgenic mice that express a constitutively active form of STAT5 (STAT5b-CA), a key downstream IL7R effector, throughout lymphocyte development. STAT5b-CA mice exhibit a 40-fold increase in pro-B cells in the thymus. As documented by BrdU labeling studies, this increase is not due to enhanced B cell proliferation. Thymic pro-B cells in STAT5b-CA mice show a modest increase in cell survival ( approximately 4-fold), which correlates with bcl-x(L) expression. However, bcl-x(L) transgenic mice do not show increases in thymic B cell numbers. Thus, STAT5-dependent bcl-x(L) up-regulation and enhanced B cell survival are not sufficient to drive the thymic B cell development observed in STAT5b-CA mice. Importantly, thymic pro-B cells in STAT5b-CA mice are derived from early T cell progenitors (ETPs), suggesting that STAT5 acts by altering ETP lineage commitment. Supporting this hypothesis, STAT5 binds to the pax5 promoter in ETPs from STAT5b-CA mice and induces pax5, a master regulator of B cell development. Conversely, STAT5b-CA mice exhibit a decrease in the DN1b subset of ETPs, demonstrating that STAT5 activation inhibits early T cell differentiation or lineage commitment. On the basis of these findings, we propose that the observed expression of the IL-7R on common lymphoid progenitors, but not ETPs, results in differential STAT5 signaling within these distinct progenitor populations and thus helps ensure appropriate development of B cells and T cells in the bone marrow and thymic environments, respectively.

STAT5 activation underlies IL7 receptor-dependent B cell development.

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R(-/-) background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R(-/-) mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-x(L) expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R(-/-) mice as determined by 1) the restoration of V(H) Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.

Distinct effects of STAT5 activation on CD4+ and CD8+ T cell homeostasis: development of CD4+CD25+ regulatory T cells versus CD8+ memory T cells.

Using transgenic mice that express a constitutively active version of STAT5b, we demonstrate that STAT5 plays a key role in governing B cell development and T cell homeostasis. STAT5 activation leads to a 10-fold increase in pro-B, but not pro-T, cells. Conversely, STAT5 signaling promotes the expansion of mature alphabeta T cells (6-fold increase) and gammadelta and NK T cells (3- to 4-fold increase), but not of mature B cells. In addition, STAT5 activation has dramatically divergent effects on CD8(+) vs CD4(+) T cells, leading to the selective expansion of CD8(+) memory-like T cells and CD4(+)CD25(+) regulatory T cells. These results establish that activation of STAT5 is the primary mechanism underlying both IL-7/IL-15-dependent homeostatic proliferation of naive and memory CD8(+) T cells and IL-2-dependent development of CD4(+)CD25(+) regulatory T cells.