A molecular dynamics approach towards evaluating osmotic and thermal stress in the extracellular environment.

OBJECTIVE: A molecular dynamics approach to understanding fundamental mechanisms of combined thermal and osmotic stress induced by thermochemical ablation (TCA) is presented. METHODS: Structural models of fibronectin and fibronectin bound to its integrin receptor provide idealized models for the effects of thermal and osmotic stress in the extracellular matrix. Fibronectin binding to integrin is known to facilitate cell survival. The extracellular environment produced by TCA at the lesion boundary was modelled at 37 °C and 43 °C with added sodium chloride (NaCl) concentrations (0, 40, 80, 160, and 320 mM). Atomistic simulations of solvated proteins were performed using the GROMOS96 force field and TIP3P water model. Computational results were compared with the results of viability studies of human hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B under matching thermal and osmotic experimental conditions. RESULTS: Cell viability was inversely correlated with hyperthermal and hyperosmotic stresses. Added NaCl concentrations were correlated with a root mean square fluctuation increase of the fibronectin arginylglycylaspartic acid (RGD) binding domain. Computed interaction coefficients demonstrate preferential hydration of the protein model and are correlated with salt-induced strengthening of hydrophobic interactions. Under the combined hyperthermal and hyperosmotic stress conditions (43 °C and 320 mM added NaCl), the free energy change required for fibronectin binding to integrin was less favorable than that for binding under control conditions (37 °C and 0 mM added NaCl). CONCLUSION: Results quantify multiple measures of structural changes as a function of temperature increase and addition of NaCl to the solution. Correlations between cell viability and stability measures suggest that protein aggregates, non-functional proteins, and less favorable cell attachment conditions have a role in TCA-induced cell stress.

Operative and Nonoperative Treatment of Humeral Shaft Fractures: A Cost-Effectiveness Analysis.

BACKGROUND: Decision-making with regard to the treatment of humeral shaft fractures remains under debate. The cost-effectiveness of these treatment options has yet to be established. This study aims to compare the cost-effectiveness of operative treatment with that of nonoperative treatment of humeral shaft fractures. METHODS: We developed a decision tree for treatment options. Surgical costs included the ambulatory surgical fee, physician fee, anesthesia fee, and, in the sensitivity analysis, lost wages during recovery. We used the Current Procedural Terminology codes from the American Board of Orthopaedic Surgery to determine physician fees via the U.S. Centers for Medicare & Medicaid Services database. The anesthesia fee was obtained from the national conversion factor and mean operative time for included procedures. We obtained data on mean wages from the U.S. Bureau of Labor and data on weeks missed from a similar study. We reported functional data via the Disabilities of the Arm, Shoulder and Hand (DASH) scores obtained from existing literature. We used rollback analysis and Monte Carlo simulation to determine the cost-effectiveness of each treatment option, presented in dollars per meaningful change in DASH score, utilizing a $50,000 willingness-to-pay (WTP) threshold. RESULTS: The cost per meaningful change in DASH score for operative treatment was $18,857.97 at the 6-month follow-up and $25,756.36 at the 1-year follow-up, by Monte Carlo simulation. Wage loss-inclusive models revealed values that fall even farther below the WTP threshold, making operative management the more cost-effective treatment option compared with nonoperative treatment in both settings. With an upward variation of the nonoperative union rate to 84.17% in the wage-exclusive model and 89.43% in the wage-inclusive model, nonoperative treatment instead became more cost-effective. CONCLUSIONS: Operative management was cost-effective at both 6 months and 1 year, compared with nonoperative treatment, in both models. Operative treatment was found to be even more cost-effective with loss of wages considered, suggesting that an earlier return to baseline function and, thus, return to work are important considerations in making operative treatment the more cost-effective option. LEVEL OF EVIDENCE: Economic and Decision Analysis Level III . See Instructions for Authors for a complete description of levels of evidence.

Rel A/p65 is required for cytokine-induced myotube atrophy.

Muscle atrophy can be triggered by systemic illnesses that are associated with elevated proinflammatory/catabolic cytokines, which, in turn, are thought to contribute to muscle wasting. In this study, we found that the prototypical NF-κB transcription factor, Rel A (p65), is required for NF-κB activation in C2C12 and L6 myotubes due to treatment with exogenous TNF-α, IL-1α, IL-1ß, TNF-related weak inducer of apoptosis, but not IL-6. All five cytokines induced atrophy in C2C12 myotubes, and inhibition of p65 reversed atrophy due to TNF-α, IL-1α, IL-1ß, TNF-related weak inducer of apoptosis, but not IL-6 treatment. p65 was also required for TNF-α-induced increase in atrophy and inflammatory gene expression. TNF-α- and IL-1ß-treated myotubes increased IL-6 protein expression, but use of an IL-6 blocking antibody showed that the IL-6 production did not contribute to atrophy. These data show that p65 is a required transcription factor mediating the catabolic effects of four different cytokines in cultured myotubes, but IL-6 works by a different mechanism.

Limb Lengthening after Primary Total Knee Arthroplasty: Customized Patient-Specific Instrumentation Does Not Affect Expected Limb Lengthening.

INTRODUCTION: Expectations for limb length differences after TKA are important for patient perception and outcomes. Limb length discrepancies may occur due to postoperative leg length increases, which can lead to decreased patient functionality and satisfaction and even possible litigation. The purpose of this study is to examine the frequency and extent of limb lengthening among various preoperative deformities and between two different implant systems. METHODS: Preoperative and postoperative full-length standing radiographs were obtained between August 2018 and August 2019 to measure mechanical axis and limb length of operative limbs. Demographic information such as age, sex, and BMI was also collected. Patients were grouped into categories for pre- and postoperative subgroup analysis: valgus, varus, customized implant, and conventional implant. Regression analysis was performed to evaluate significant relationships. RESULTS: Of the 121 primary TKAs analyzed, 62% of the knees showed an increase in limb length after TKA, with an average lengthening of 5.32 mm. Preoperative varus alignment was associated with a mean lengthening of 3.14 mm, while preoperative valgus alignment was associated with a mean lengthening of 16.2 mm. Overall, there were no statistically significant differences in limb lengths pre- and postoperatively (p = 0.23) and no significant changes in limb length for any subgroup. Further, no variables were associated with limb length changes (p = 0.49), including the use of customized implants (p = 0.2). CONCLUSIONS: Limb lengthening after TKA is common and, on average, occurs more significantly in valgus knees. No significant difference in limb lengthening could be demonstrated using customized over conventional implants. Preoperative counseling is important to manage patient expectations.

The Modified Eden-Lange Tendon Transfer for Lateral Scapular Winging Secondary to Spinal Accessory Nerve Injury.

Trapezius paralysis following injury to the spinal accessory nerve can be a debilitating complication resulting from lymph node biopsy, radical neck dissection, or penetrating trauma in the region of the posterior cervical triangle. Disruption of the delicate muscular balance in the shoulder girdle may result in lateral scapular winging, ipsilateral upper extremity radiculopathy, and limited shoulder function and range of motion. Spontaneous recovery with nonoperative management is possible in some patients, and restoration of function after reparative neural procedures has been observed in patients undergoing timely repair. However, extended delays from the time of injury to surgery are common and may necessitate various muscle transfers to reestablish the complex biomechanics and balance of the shoulder girdle. We describe a modification to the classic Eden-Lange procedure with lateral transfer of the levator scapulae and rhomboid minor to the scapula spine and rhomboid major transfer with a small wafer of bone to the scapula body for chronic lateral winging of the scapula following injury to the spinal accessory nerve as the result of a cervical lymph node biopsy.

Return to Sport After ACL Reconstruction With a BTB Versus Hamstring Tendon Autograft: A Systematic Review and Meta-analysis.

BACKGROUND: Anterior cruciate ligament (ACL) tears are debilitating injuries frequently suffered by athletes. ACL reconstruction is indicated to restore knee stability and allow patients to return to prior levels of athletic performance. While existing literature suggests that patient-reported outcomes are similar between bone-patellar tendon-bone (BTB) and hamstring tendon (HT) autografts, there is less information comparing return-to-sport (RTS) rates between the 2 graft types. PURPOSE: To compare RTS rates among athletes undergoing primary ACL reconstruction using a BTB versus HT autograft. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: The MEDLINE, Embase, and Cochrane Library databases were searched, and studies that reported on RTS after primary ACL reconstruction using a BTB or HT autograft were included. Studies that utilized ACL repair techniques, quadriceps tendon autografts, graft augmentation, double-bundle autografts, allografts, or revision ACL reconstruction were excluded. RTS information was extracted and analyzed from all included studies. RESULTS: Included in the review were 20 articles investigating a total of 2348 athletes. The overall RTS rate in our cohort was 73.2%, with 48.9% returning to preinjury levels of performance and a rerupture rate of 2.4%. The overall RTS rate in patients after primary ACL reconstruction with a BTB autograft was 81.0%, with 50.0% of athletes returning to preinjury levels of performance and a rerupture rate of 2.2%. Patients after primary ACL reconstruction with an HT autograft had an overall RTS rate of 70.6%, with 48.5% of athletes returning to preinjury levels of performance and a rerupture rate of 2.5%. CONCLUSION: ACL reconstruction using BTB autografts demonstrated higher overall RTS rates when compared with HT autografts. However, BTB and HT autografts had similar rates of return to preinjury levels of performance and rerupture rates. Less than half of the athletes were able to return to preinjury sport levels after ACL reconstruction with either an HT or BTB autograft.

CTA4 transcription factor mediates induction of nitrosative stress response in Candida albicans.

This work has identified regulatory elements in the major fungal pathogen Candida albicans that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of C. albicans inside the human body. Exposing C. albicans to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how C. albicans is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the YHB1 regulatory region. Deletion of CTA4 caused a small but significant decrease in virulence. A CTA4-dependent putative sulfite transporter encoded by SSU1 is also implicated in NO response, but C. albicans ssu1 mutants were not sensitive to NO, in contrast to findings in Saccharomyces cerevisiae. Cta4p is the first protein found to be necessary for initiating NO response in C. albicans.

Management of Complex Anterior Shoulder Instability: a Case-Based Approach.

PURPOSE OF THE REVIEW: The goal of this review is to provide a guide on surgical decision-making options for complex anterior shoulder instability using a case-based approach. RECENT FINDINGS: Arthroscopic Bankart repair is well documented for having successful outcomes in patients with isolated labral tear involvement with minimal bone loss. Latarjet is a generally accepted procedure in patients with 20-30% glenoid bone loss. When bone loss exceeds that which cannot be managed through Latarjet, a range of options exist and are highly dependent upon the extent of osseous deficiency on both the glenoid and humeral sides, surgeon experience, and patient-specific factors. The use of reverse total shoulder arthroplasty for the management of chronic locked shoulder dislocations has been described as a successful management option. Treatment options for complex anterior shoulder instability range widely based on patients' presenting exam, surgical history, amount of glenoid bone loss, size of Hill-Sachs lesion, and surgeon preference. When selecting the appropriate surgical intervention, the treating surgeon must consider the patient history, physical exam, and preoperative imaging along with patient expectations.

Adaptation of Candida albicans to Reactive Sulfur Species.

Candida albicans is an opportunistic fungal pathogen that is highly resistant to different oxidative stresses. How reactive sulfur species (RSS) such as sulfite regulate gene expression and the role of the transcription factor Zcf2 and the sulfite exporter Ssu1 in such responses are not known. Here, we show that C. albicans specifically adapts to sulfite stress and that Zcf2 is required for that response as well as induction of genes predicted to remove sulfite from cells and to increase the intracellular amount of a subset of nitrogen metabolites. Analysis of mutants in the sulfate assimilation pathway show that sulfite conversion to sulfide accounts for part of sulfite toxicity and that Zcf2-dependent expression of the SSU1 sulfite exporter is induced by both sulfite and sulfide. Mutations in the SSU1 promoter that selectively inhibit induction by the reactive nitrogen species (RNS) nitrite, a previously reported activator of SSU1, support a model for C. albicans in which Cta4-dependent RNS induction and Zcf2-dependent RSS induction are mediated by parallel pathways, different from S. cerevisiae in which the transcription factor Fzf1 mediates responses to both RNS and RSS. Lastly, we found that endogenous sulfite production leads to an increase in resistance to exogenously added sulfite. These results demonstrate that C. albicans has a unique response to sulfite that differs from the general oxidative stress response, and that adaptation to internal and external sulfite is largely mediated by one transcription factor and one effector gene.

The Drosophila inebriated-encoded neurotransmitter/osmolyte transporter: dual roles in the control of neuronal excitability and the osmotic stress response.

Water reabsorption by organs such as the mammalian kidney and insect Malpighian tubule/hindgut requires a region of hypertonicity within the organ. To balance the high extracellular osmolarity, cells within these regions accumulate small organic molecules called osmolytes. These osmolytes can accumulate to a high level without toxic effects on cellular processes. Here we provide evidence consistent with the possibility that the two protein isoforms encoded by the inebriated (ine) gene, which are members of the Na+/Cl--dependent neurotransmitter/osmolyte transporter family, perform osmolyte transport within the Malpighian tubule and hindgut. We show that ine mutants lacking both isoforms are hypersensitive to osmotic stress, which we assayed by maintaining flies on media containing NaCl, KCl, or sorbitol, and that this hypersensitivity is completely rescued by high-level ectopic expression of the ine-RB isoform. We provide evidence that this hypersensitivity represents a role for ine that is distinct from the increased neuronal excitability phenotype of ine mutants. Finally, we show that each ine genotype exhibits a "threshold" [NaCl]: long-term maintenance on NaCl-containing media above, but not below, the threshold causes lethality. Furthermore, this threshold value increases with the amount of ine activity. These data suggest that ine mutations confer osmotic stress sensitivity by preventing osmolyte accumulation within the Malpighian tubule and hindgut.

Hog1: 20 years of discovery and impact.

The protein kinase Hog1 (high osmolarity glycerol 1) was discovered 20 years ago, being revealed as a central signaling mediator during osmoregulation in the budding yeast Saccharomyces cerevisiae. Homologs of Hog1 exist in all evaluated eukaryotic organisms, and this kinase plays a central role in cellular responses to external stresses and stimuli. Here, we highlight the mechanism by which cells sense changes in extracellular osmolarity, the method by which Hog1 regulates cellular adaptation, and the impacts of the Hog1 pathway upon cellular growth and morphology. Studies that have addressed these issues reveal the influence of the Hog1 signaling pathway on diverse cellular processes.

From START to FINISH: the influence of osmotic stress on the cell cycle.

The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle of S. cerevisiae for the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments in S. cerevisiae and, moreover, have recently been observed in other eukaryotes.

The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9.

Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17 Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel ß-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones.

MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells.

Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) --> MAPK kinase (MKK) --> MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 -->--> p38 kinase -->--> TonE.

Imidazole antibiotics inhibit the nitric oxide dioxygenase function of microbial flavohemoglobin.

Flavohemoglobins metabolize nitric oxide (NO) to nitrate and protect bacteria and fungi from NO-mediated damage, growth inhibition, and killing by NO-releasing immune cells. Antimicrobial imidazoles were tested for their ability to coordinate flavohemoglobin and inhibit its NO dioxygenase (NOD) function. Miconazole, econazole, clotrimazole, and ketoconazole inhibited the NOD activity of Escherichia coli flavohemoglobin with apparent K(i) values of 80, 550, 1,300, and 5,000 nM, respectively. Saccharomyces cerevisiae, Candida albicans, and Alcaligenes eutrophus enzymes exhibited similar sensitivities to imidazoles. Imidazoles coordinated the heme iron atom, impaired ferric heme reduction, produced uncompetitive inhibition with respect to O(2) and NO, and inhibited NO metabolism by yeasts and bacteria. Nevertheless, these imidazoles were not sufficiently selective to fully mimic the NO-dependent growth stasis seen with NOD-deficient mutants. The results demonstrate a mechanism for NOD inhibition by imidazoles and suggest a target for imidazole engineering.

MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals.

The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2. In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity. This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals.

Inducible defense mechanism against nitric oxide in Candida albicans.

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.