Automated Platform for the Plasmid Construction Process.

There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and manipulating DNA as plasmids. Here, we report the development of an application named DNAda capable of writing automation instructions for any given DNA construct design generated by the J5 DNA assembly program. We also describe the automation pipeline and several useful features. The pipeline is particularly useful for the construction of combinatorial DNA assemblies. Furthermore, we demonstrate the platform by constructing a library of polyketide synthase parts, which includes 120 plasmids ranging in size from 7 to 14 kb from 4 to 7 DNA fragments.

Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format.

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes (e.g., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5-2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

Nasal Sesamoid Cartilages: Fact or Fiction? An Anatomical Study of the Lateral Nasal Ala.

OBJECTIVES: To evaluate the presence of cartilage in the lateral nasal ala (LNA) via histological analysis of alar Mohs surgical specimens. An accurate understanding of nasal anatomy is essential to optimize esthetic and reconstructive surgical outcomes. LNA anatomy is typically pictured to include discreet accessory and sesamoid cartilages; however, the authors debate the actual presence of these structures. METHODS: A blinded reviewer analyzed 101 lateral nasal alar histologic specimens from 362 tissue blocks using a Hematoxylin and Eosin (H&E) stain to assess for the presence of cartilage. RESULTS: Out of the 362 tissue blocks histologically analyzed, only 1 included cartilaginous components. CONCLUSIONS: We conclude that the presence of sesamoid and accessory cartilages in the LNA within our sample set is exceedingly rare.

Actigraphic and nursing sleep log measures in moderate-to-severe traumatic brain injury: Identifying discrepancies in total sleep time.

BACKGROUND: Sleep disturbances are common in patients with traumatic brain injury (TBI). In an inpatient rehabilitation setting, clinicians often use information from sleep logs filled out by trained nurses to identify and treat sleep disturbances. However, there are limited data related to accuracy of sleep logs, and patient-reported sleep diaries are poor predictors of total sleep time, which raises concern about the accuracy of sleep logs filled out by a third party. OBJECTIVE: To examine the reliability of sleep logs for participants with TBI by comparing total sleep time determined by sleep logs versus actigraphy. DESIGN: Prospective, cross-sectional study. SETTING: Free-standing, academic inpatient rehabilitation facility. PARTICIPANTS: Thirty individuals (n = 30) participated in the study. Inclusion criteria were (1) diagnosis of moderate-to-severe TBI; (2) age ≥ 18 years at the time of TBI; and (3) participating in inpatient rehabilitation with no prior inpatient rehabilitation admissions. INTERVENTIONS: Actigraph monitoring using ActiGraph GT9X Link devices was initiated within 72 hours of admission and continued for 7 consecutive days. Sleep logs were concurrently filled out by trained nurses. MAIN OUTCOME MEASURES: Sleep parameter correspondence between actigraphy and sleep logs in moderate-to-severe TBI. RESULTS: Only 51.4% of participants' sleep logs and actigraph total sleep time measurements were within 1 hour of each other, and only 23.8% were within 30 minutes. On average, sleep logs overestimated actigraphy-determined total sleep time by 60 minutes compared to actigraphic measurement. CONCLUSIONS: For those with moderate-to-severe TBI undergoing inpatient rehabilitation, sleep logs are poor predictors of sleep time because they overestimate total sleep time compared to actigraphy. Therefore, clinicians should use caution when using sleep log data to make decisions regarding treatment for sleep disturbances in TBI.

Data Velocity in HIV-Related Implementation Research: Estimating Time From Funding to Publication.

BACKGROUND: Given available effective biomedical and behavioral prevention and treatment interventions, HIV-related implementation research (IR) is expanding. The rapid generation and dissemination of IR to inform guidelines and practice has the potential to optimize the impact of the Ending the Epidemic Initiative and the HIV pandemic response more broadly. METHODS: We leveraged a prior mapping review of NIH-funded awards in HIV and IR from January 2013 to March 2018 and identified all publications linked to those grants in NIH RePORTER through January 1, 2021 (n = 1509). Deduplication and screening of nonoriginal research reduced the count to 1032 articles, of which 952 were eligible and included in this review. Publication volume and timing were summarized; Kaplan-Meier plots estimated time to publication. RESULTS: Among the 215 NIH-funded IR-related awards, 127 of 215 (59%) published original research directly related to the grant, averaging 2.0 articles (SD: 3.3) per award, largely in the early IR phases. Many articles (521 of 952, 55%) attributed to grants did not report grant-related data. Time from article submission to publication averaged 205 days (SD: 107). The median time-to-first publication from funding start was 4 years. Data dissemination velocity varied by award type, trending toward faster publication in recent years. Delays in data velocity included (1) time from funding to enrollment, (2) enrollment length, and (3) time from data collection completion to publication. CONCLUSION: Research publication was high overall, and time-to-publication is accelerating; however, over 40% of grants have yet to publish findings from grant-related data. Addressing bottlenecks in the production and dissemination of HIV-related IR would reinforce its programmatic and policy relevance in the HIV response.

Cortical Transplantation of Brain-Mimetic Glycosaminoglycan Scaffolds and Neural Progenitor Cells Promotes Vascular Regeneration and Functional Recovery after Ischemic Stroke in Mice.

Stroke causes significant mortality and morbidity. Currently, there are no treatments which can regenerate brain tissue lost to infarction. Neural progenitor cells (NPCs) are at the forefront of preclinical studies for regenerative stroke therapies. NPCs can differentiate into and replace neurons and promote endogenous recovery mechanisms such as angiogenesis via trophic factor production and release. The stroke core is hypothetically the ideal location for replacement of neural tissue since it is in situ and develops into a potential space where injections may be targeted with minimal compression of healthy peri-infarct tissue. However, the compromised perfusion and tissue degradation following ischemia create an inhospitable environment resistant to cellular therapy. Overcoming these limitations is critical to advancing cellular therapy. In this work, the therapeutic potential of mouse-induced pluripotent stem cell derived NPCs is tested encapsulated in a basic fibroblast growth factor (bFGF) binding chondroitin sulfate-A (CS-A) hydrogel transplanted into the infarct core in a mouse sensorimotor cortex mini-stroke model. It is shown that CS-A encapsulation significantly improves vascular remodeling, cortical blood flow, and sensorimotor behavioral outcomes after stroke. It is found these improvements are negated by blocking bFGF, suggesting that the sustained trophic signaling endowed by the CS-A hydrogel combined with NPC transplantation can promote tissue repair.

Infant Trauma Alters Social Buffering of Threat Learning: Emerging Role of Prefrontal Cortex in Preadolescence.

Within the infant-caregiver attachment system, the primary caregiver holds potent reward value to the infant, exhibited by infants' strong preference for approach responses and proximity-seeking towards the mother. A less well-understood feature of the attachment figure is the caregiver's ability to reduce fear via social buffering, commonly associated with the notion of a "safe haven" in the developmental literature. Evidence suggests this infant system overlaps with the neural network supporting social buffering (attenuation) of fear in the adults of many species, a network known to involve the prefrontal cortex (PFC). Here, using odor-shock conditioning in young developing rats, we assessed when the infant system transitions to the adult-like PFC-dependent social buffering of threat system. Rat pups were odor-shock conditioned (0.55 mA-0.6 mA) at either postnatal day (PN18; dependent on mother) or 28 (newly independent, weaned at PN23). Within each age group, the mother was present or absent during conditioning, with PFC assessment following acquisition using 14C 2-DG autoradiography and cue testing the following day. Since the human literature suggests poor attachment attenuates the mother's ability to socially buffer the infants, half of the pups at each age were reared with an abusive mother from PN8-12. The results showed that for typical control rearing, the mother attenuated fear in both PN18 and PN28 pups, although the PFC [infralimbic (IL) and ventral prelimbic (vPL) cortices] was only engaged at PN28. Abuse rearing completely disrupted social buffering of pups by the mother at PN18. The results from PN28 pups showed that while the mother modulated learning in both control and abuse-reared pups, the behavioral and PFC effects were attenuated after maltreatment. Our data suggest that pups transition to the adult-like PFC social support circuit after independence from the mother (PN28), and this circuit remains functional after early-life trauma, although its effectiveness appears reduced. This is in sharp contrast to the effects of early life trauma during infancy, where social buffering of the infant is more robustly impacted. We suggest that the infant social buffering circuit is disengaged by early-life trauma, while the adolescent PFC-dependent social buffering circuit may use a safety signal with unreliable safety value.

Neurobiology of maternal regulation of infant fear: the role of mesolimbic dopamine and its disruption by maltreatment.

Child development research highlights caregiver regulation of infant physiology and behavior as a key feature of early life attachment, although mechanisms for maternal control of infant neural circuits remain elusive. Here we explored the neurobiology of maternal regulation of infant fear using neural network and molecular levels of analysis in a rodent model. Previous research has shown maternal suppression of amygdala-dependent fear learning during a sensitive period. Here we characterize changes in neural networks engaged during maternal regulation and the transition to infant self-regulation. Metabolic mapping of 2-deoxyglucose uptake during odor-shock conditioning in postnatal day (PN)14 rat pups showed that maternal presence blocked fear learning, disengaged mesolimbic circuitry, basolateral amygdala (BLA), and plasticity-related AMPA receptor subunit trafficking. At PN18, when maternal presence only socially buffers threat learning (similar to social modulation in adults), maternal presence failed to disengage the mesolimbic dopaminergic system, and failed to disengage both the BLA and plasticity-related AMPA receptor subunit trafficking. Further, maternal presence failed to block threat learning at PN14 pups following abuse, and mesolimbic dopamine engagement and AMPA were not significantly altered by maternal presence-analogous to compromised maternal regulation of children in abusive relationships. Our results highlight three key features of maternal regulation: (1) maternal presence blocks fear learning and amygdala plasticity through age-dependent suppression of amygdala AMPA receptor subunit trafficking, (2) maternal presence suppresses engagement of brain regions within the mesolimbic dopamine circuit, and (3) early-life abuse compromises network and molecular biomarkers of maternal regulation, suggesting reduced social scaffolding of the brain.

Clinical outcomes and hospital length of stay in 2,756 elderly patients with hip fractures: a comparison of surgical and non-surgical management.

INTRODUCTION: The purpose of this study was to compare the clinical outcomes of elderly hip fracture patients who received surgical treatment with those who received non-surgical treatment. METHODS: This retrospective study involved 2,756 elderly patients with hip fractures who were admitted over a six-year period. The patients' biodata, complications, ambulatory status at discharge and length of hospital stay were obtained from the institution's hip fracture registry. RESULTS: Among the 2,756 hip fracture patients, 2,029 (73.6%) underwent surgical intervention, while 727 (26.4%) opted for non-surgical intervention. The complication rate among the patients who underwent surgical intervention was 6.6%, while that among the patients who underwent non-surgical intervention was 12.5% (p < 0.01). The mean length of hospital stay for the surgical and non-surgical hip fracture patients was 15.7 days and 22.4 days, respectively (p < 0.01). CONCLUSION: Surgical management of hip fractures among the elderly is associated with a lower complication rate, as well as a reduced length of hospital stay.

Short-course of ranolazine prevents postoperative atrial fibrillation following coronary artery bypass grafting and valve surgeries.

BACKGROUND: Postoperative atrial fibrillation (POAF) is a common complication arising after coronary artery bypass grafting (CABG) and valve replacement or repair surgeries. POAF has been associated with increased mortality, morbidity and cost. METHODS: The study was conducted to evaluate the incidence of POAF following CABG, valve or combination surgeries when perioperative ranolazine (1,000 mg preoperatively, then 1,000 mg twice daily for 7 days or until discharge) was or was not added to standard therapy. RESULTS: A total of 205 patients were evaluated for POAF after CABG, valve or combination surgeries. POAF occurred less frequently in the ranolazine group compared with the non-ranolazine group in unmatched analysis (10.1 vs. 41.9 %, p < 0.0001). After adjusting for potential sources of bias through propensity-score matched-pair analysis and conditional logistic regression, ranolazine was an independent predictor of preventing POAF (p < 0.0001). There were no differences in bradycardia, new renal failure or neurological events between the two groups. Early, symptomatic hypotension occurred more frequently in the ranolazine group (p = 0.0004) although this difference did not persist after 72 h. No significant difference was found in the length of stay in the intensive care unit following cardiac surgery. While a significant difference was found in the hospital readmission rate for a cardiac cause within 30 days in the unmatched analysis (p = 0.046), this difference was nonexistent after matching (p = 0.39). No difference was found in 30-day cardiovascular mortality. CONCLUSION: Adding ranolazine to standard therapy was independently associated with a significant decrease in POAF development after CABG, valve or combination surgeries.

Association between type of reconstruction after Mohs micrographic surgery and surgeon-, patient-, and tumor-specific features: a cross-sectional study.

BACKGROUND: There are few data to indicate whether the type of final wound defect is associated with the type of post-Mohs repair. OBJECTIVE: To determine the methods of reconstruction that Mohs surgeons typically select and, secondarily, to assess the association between the method and the number of stages, tumor type, anatomic location, and patient and surgeon characteristics. METHODS: Statistical analysis of procedure logs of 20 representative young to mid-career Mohs surgeons. RESULTS: The number of stages associated with various repairs were different (analysis of variance, p < .001.). Linear repairs, associated with the fewest stages (1.5), were used most commonly (43-55% of defects). Primary repairs were used for 20.2% to 35.3% of defects of the nose, eyelids, ears, and lips. Local flaps were performed typically after two stages of Mohs surgery (range 1.98-2.06). Referral for repair and skin grafts were associated with cases with more stages (2.16 and 2.17 stages, respectively). Experienced surgeons were nominally more likely perform flaps than grafts. Regression analyses did not indicate any association between patient sex and closure type (p = .99) or practice location and closure type (p = .99). CONCLUSIONS: Most post-Mohs closures are linear repairs, with more bilayered linear repairs more likely at certain anatomic sites and after a larger number of stages.

Nutrients and suspended solids in dry weather and storm flows from a tropical catchment with various proportions of rural and urban land use.

The results of an investigation characterizing the nutrients and suspended solids contained in stormwater from Kranji Catchment in Singapore are reported in this paper. Stormwater samples were collected from 4 locations and analyzed for the following eleven analytes: TOC, DOC, TN, TDN, NH(4)(+), NO(2)(-)+NO(3)(-) (NO(x)), TP, TDP, OP, SiO(2) and TSS. Stormwater was sampled from catchments with various proportions of rural and urban land use, including forested areas, grassed areas, agricultural and residential and commercial areas. The event mean concentrations (EMCs) of nutrients and TSS from sampling stations which have agricultural land use activities upstream were found to be higher. Comparison of site EMCs (SMCs) with published data showed that the SMCs of the nutrients and TSS are generally higher than SMCs reported for forested areas but lower than published SMCs for urban areas. Positive correlations (p<5%) were found between loading and peak flow at locations most impacted by ubanisation or agricultural activities. Correlation between loading and rainfall variables was less distinct. EMC was found to correlate less with rainfall and flow variables compared to pollutant loading. Unlike loading, no consistent pattern exists linking EMC to any particular storm or flow variable in any of the catchments. Lastly, positive correlations were obtained between the particulate forms of nitrogen and phosphorus and TSS.

Pemphigus vulgaris induced by electrical injury.

Pemphigus refers to a group of autoimmune blistering diseases that affect the skin and mucous membranes. Pemphigus may be induced following exposure to various exogenous agents, including thermal burns, drugs, infectious agents, and neoplasms, as well as UV, ionizing, and x-ray irradiation. We report a case of a 28-year-old man with pemphigus vulgaris (PV) induced by a severe electrical injury. Approximately one month after the electrical injury, he began to develop recurrent painful oral ulcers; one year later, he began to develop cutaneous bullae. Results of a histopathologic examination and immunofluorescence studies were diagnostic of PV The primary mechanisms of high-voltage electrical injury involve electroporation, electroconformational protein denaturation, and both joule and dielectric heating. Cutaneous electrical injury ultimately results in the destruction of cells with release of their cellular constituents. Through these mechanisms, desmoglein 3 (Dsg3) may be released and become available to the immune system, which potentially leads to an autoantibody response and the subsequent development of PV.

Pulsed dye laser treatment of rosacea improves erythema, symptomatology, and quality of life.

BACKGROUND: Persistent erythema and dysesthetic symptoms are typical manifestations of rosacea. OBJECTIVE: We sought to assess improvement in erythema, symptoms, and quality of life after pulsed dye laser treatment. METHODS: Sixteen patients with erythematotelangiectatic rosacea participated. Spectrophotometric erythema measurements were taken from the right and left malar prominence; chin; and nasal alae, dorsum, and tip. A questionnaire rating the Dermatology Life Quality Index and symptoms of flushing, burning, itching, dryness, swelling, and skin sensitivity was completed. Treatment was undertaken with the pulsed dye laser at purpuragenic fluences. Measurements and treatment were repeated at 8-week intervals for a total of two treatments. RESULTS: A statistically significant improvement was observed in symptoms, quality-of-life score, and erythema in all areas with the exception of erythema of the left nasal ala. CONCLUSIONS: Pulsed dye laser treatment at purpuragenic fluences is a safe and effective treatment for symptomatic rosacea, resulting in a significant improvement in erythema, symptoms, and quality of life.

Effectiveness of microporous polysaccharide hemospheres for achieving hemostasis in mohs micrographic surgery.

BACKGROUND: Microporous polysaccharide hemospheres consist of controlled-porosity spherical particles manufactured from bioinert plant polysaccharide. Microporous polysaccharide hemospheres facilitate hemostasis by rapidly absorbing the fluid component of blood, concentrating platelets and clotting factors to accelerate blood clotting. OBJECTIVE: The objective was to compare a microporous polysaccharide hemosphere bandage and electrocautery in achieving hemostasis. METHODS: Twenty-four patients with a total of 48 stages of Mohs micrographic surgery were included. Patients were stratified by whether or not they were taking anticoagulant medications. Within each group, patients were randomized to receive either the microporous polysaccharide hemosphere bandage or electrocautery. Outcomes included bleeding through the dressing (early time point) and active bleeding upon dressing removal (late time point). RESULTS: Nineteen patients not taking anticoagulants had 40 stages, of which 18 received the study bandage and 22 received electrocautery. The remaining 5 patients on anticoagulants had 8 stages, of which 4 received the study bandage and 4 received electrocautery. In both total and subgroup analysis, there was a higher incidence of bleeding through the dressing with the study bandage (p<0.05), but no increase in the incidence of active bleeding upon dressing removal (p>0.05). CONCLUSION: The microporous polysaccharide hemosphere study bandage had an increased incidence of bleeding through the dressing compared to electrocautery, but did not have an increased incidence of active bleeding upon dressing removal.

Effect of acitretin on wound healing in organ transplant recipients.

BACKGROUND: Systemic retinoids possess significant benefits in cutaneous malignancy chemoprevention; however, retinoids have been associated with excessive granulation tissue and hypertrophic scarring. OBJECTIVE: The objective of this study was to assess wound healing outcomes in organ transplant recipients, both with and without the concomitant use of systemic acitretin chemoprophylaxis. METHODS: Twenty-nine immunosuppressed organ transplant recipients underwent treatment of basal cell or squamous cell carcinoma by Mohs or excisional surgery, with a total of 85 wounds. Wounds were evaluated postoperatively at early (average 12.9 days) and late (average 75.8 days) time points. Endpoints for all wounds included infection, hypertrophic granulation tissue, and hypertrophic scarring. Reconstructed wounds were also evaluated for dehiscence. RESULTS: Eleven patients taking oral acitretin had 41 wounds, of which 33 were reconstructed and 8 healed by second intention. The 18 patients not taking acitretin (control group) had a total of 44 wounds, of which 33 were reconstructed and 11 healed by second intention. There were no statistically significant differences between the acitretin group and the control group in the incidences of infection, dehiscence, hypertrophic granulation tissue, or hypertrophic scarring at early or late evaluation points. CONCLUSIONS: Systemic acitretin chemoprophylaxis does not appear to increase the risk of wound healing complications in organ transplant recipients.

Generalized cutis laxa associated with heavy chain deposition disease.

BACKGROUND: Cutis laxa is a heterogeneous group of inherited and acquired disorders characterized clinically by loose skin and histologically by altered elastic tissue. Heavy chain deposition disease is a very rare monoclonal immunoglobulin disorder, distinct from multiple myeloma, in which there is production and deposition of defective immunoglobulin heavy chains without light chain deposition. OBJECTIVE: We describe a case of acquired cutis laxa associated with heavy chain deposition disease. RESULTS: A 50-year-old male presented with acute renal failure, IgG4 heavy chain deposition in the kidneys, and no evidence of multiple myeloma. Four years later, he developed generalized acquired cutis laxa, emphysema, and a peripheral polyneuropathy. On pathology, there was destruction of elastic fibers within the dermis. CONCLUSION: This case describes a previously unreported association between acquired cutis laxa and heavy chain deposition disease.