The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3'UTR of CPSF6 contains a miR-125b-binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3'UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3'UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3'UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3'UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

Q-plates with a nonlinear azimuthal distribution of the principal axis: application to encoding binary data.

We encode q-plates where the angular orientation of the principal axis is varied spatially with a nonstandard distribution. In the usual q-plate design, the orientation of the optical axis depends linearly on the azimuthal angle. In this work, we examine cases where this azimuthal dependence is nonlinear. We consider two cases: first, where the principal axis distribution is like an inverse-tangent function of the azimuth; and second, where it displays linear and flat segments. This last case is proposed as a new method for encoding binary data into the azimuthal lobes of the vector beam. We encode these patterns using a spatial light modulator system that allows new and exotic q-plate designs without the difficulty of fabricating individual plates. Experimental results are presented.

Evaluating the Feasibility of a Novel Firearm Injury Prevention Program for Pre-adolescent Children Through Health Care and Community-Based Partnerships: The Future Healers Program Pilot Study.

BACKGROUND: Firearm violence is an American public health crisis that negatively impacts children and disproportionately affects Black youth. Few firearm injury prevention programs have been described in pre-adolescent children. The Future Healers Program is a novel collaboration constructed via partnership between the medical school, trauma center, academic surgery department, and local non-profit community organization. Our study sought to evaluate if (1) partnering with community organizations facilitated recruitment of children with prior exposure to firearm violence and (2) the health care community was a potential trusted partner appropriate for program delivery. METHODS: Children aged 4-13 were recruited to join the program via news outlets and social media and in partnership with a local non-profit organization. Of the children and parents participating in the program, 48% (44/92) and 59% (38/64), respectively, completed an IRB-approved survey study. Pearson's chi-square, percentages, and 95% confidence intervals evaluated differences between children and caregivers on sociodemographic characteristics, firearm exposure (FE), firearm violence exposure (FVE), and perception of health care. Participant's residence was geocoded in relationship to incidents of firearm injury (2008-2021) in the same region. RESULTS: Caregivers (95%) and children (84%) reported substantial exposure to firearm violence and resided in areas with frequent firearm injury incidents. Notably, 82% of caregivers and 66% of children reported having a family member injured by gunfire. A high percentage of caregivers (79%) and children (91%) self-reported trust in the health care system. CONCLUSION: Partnerships between community organizations and health care systems can develop prevention programs that effectively recruit and engage pre-adolescent children impacted by firearm violence.

Counting the number of magnesium ions bound to the surface-immobilized thymine oligonucleotides that comprise spherical nucleic acids.

Label-free studies carried out under aqueous phase conditions quantify the number of Mg(2+) ions binding to surface-immobilized T40 sequences, the subsequent reordering of DNA on the surface, and the consequences of Mg(2+) binding for DNA-DNA interactions. Second harmonic generation measurements indicate that, within error, 18-20 Mg(2+) ions are bound to the T40 strand at saturation and that the metal-DNA interaction is associated with a near 30% length contraction of the strand. Structural reordering, evaluated using vibrational sum frequency generation, atomic force microscopy, and dynamic light scattering, is attributed to increased charge screening as the Mg(2+) ions bind to the negatively charged DNA, reducing repulsive Coulomb forces between nucleotides and allowing the DNA single strands to collapse or coil upon themselves. The impact of Mg(2+) binding on DNA hybridization and duplex stability is assessed with spherical nucleic acid (SNA) gold nanoparticle conjugates in order to determine an optimal working range of Mg(2+) concentrations for DNA-DNA interactions in the absence of NaCl. The findings are consistent with a charge titration effect in which, in the absence of NaCl, (1) hybridization does not occur at room temperature if an average of 17.5 or less Mg(2+) ions are bound per T40 strand, which is not reached until the bulk Mg(2+) concentration approaches 0.5 mM; (2) hybridization proceeds, albeit with low duplex stability having an average Tm of 31(3)°C, if an average of 17.5-18.0 Mg(2+) ions are bound; and (3) highly stable duplexes having a Tm of 64(2)°C form if 18.5-19.0 Mg(2+) ions are bound, corresponding to saturation of the T40 strand.

Sociodemographic Characteristics of Patients Undergoing Surgery for Metastatic Disease of the Spine.

INTRODUCTION: Some patients, particularly those who are socioeconomically deprived, are diagnosed with primary and/or metastatic cancer only after presenting to the emergency department. Our objective was to determine sociodemographic characteristics of patients undergoing surgery for metastatic spine disease at our institution. METHODS: This retrospective case series included patients 18 years and older who presented to the emergency department with metastatic spine disease requiring surgery. Demographics and survival data were collected. Sociodemographic characteristics were estimated using the Social Deprivation Index (SDI) and Area Deprivation Index (ADI) for the state of California. Univariate log-rank tests and Kaplan-Meier curves were used to assess differences in survival for predictors of interest. RESULTS: Between 2015 and 2021, 64 patients underwent surgery for metastatic disease of the spine. The mean age was 61.0 ± 12.5 years, with 60.9% being male (n = 39). In this cohort, 89.1% of patients were non-Hispanic (n = 57), 71.9% were White (n = 46), and 62.5% were insured by Medicare/Medicaid (n = 40). The mean SDI and ADI were 61.5 ± 28.0 and 7.7 ± 2.2, respectively. 28.1% of patients (n = 18) were diagnosed with primary cancer for the first time while 39.1% of patients (n = 25) were diagnosed with metastatic cancer for the first time. During index hospitalization, 37.5% of patients (n = 24) received palliative care consult. The 3-month, 6-month, and all-time mortality rates were 26.7% (n = 17), 39.5% (n = 23), and 50% (n = 32), respectively, with 10.9% of patients (n = 7) dying during their admission. Payor plan was significant at 3 months ( P = 0.02), and palliative consultation was significant at 3 months ( P = 0.007) and 6 months ( P = 0.03). No notable association was observed with SDI and ADI in quantiles or as continuous variables. DISCUSSION: In this study, 28.1% of patients were diagnosed with cancer for the first time. Three-month and 6-month mortality rates for patients undergoing surgery were 26.7% and 39.5%, respectively. Furthermore, mortality was markedly associated with palliative care consultation and insurance status, but not with SDI and ADI. LEVEL OF EVIDENCE: Retrospective case series, Level III evidence.

A Comparison of Prognostic Models to Facilitate Surgical Decision-Making for Patients With Spinal Metastatic Disease.

STUDY DESIGN: Retrospective cohort. OBJECTIVE: Compare the performance of and provide cutoff values for commonly used prognostic models for spinal metastases, including Revised Tokuhashi, Tomita, Modified Bauer, New England Spinal Metastases Score (NESMS), and Skeletal Oncology Research Group model, at three- and six-month postoperative time points. SUMMARY OF BACKGROUND DATA: Surgery may be recommended for patients with spinal metastases causing fracture, instability, pain, and/or neurological compromise. However, patients with less than three to six months of projected survival are less likely to benefit from surgery. Prognostic models have been developed to help determine prognosis and surgical candidacy. Yet, there is a lack of data directly comparing the performance of these models at clinically relevant time points or providing clinically applicable cutoff values for the models. MATERIALS AND METHODS: Sixty-four patients undergoing surgery from 2015 to 2022 for spinal metastatic disease were identified. Revised Tokuhashi, Tomita, Modified Bauer, NESMS, and Skeletal Oncology Research Group were calculated for each patient. Model calibration and discrimination for predicting survival at three months, six months, and final follow-up were evaluated using the Brier score and Uno's C, respectively. Hazard ratios for survival were calculated for the models. The Contral and O'Quigley method was utilized to identify cutoff values for the models discriminating between survival and nonsurvival at three months, six months, and final follow-up. RESULTS: Each of the models demonstrated similar performance in predicting survival at three months, six months, and final follow-up. Cutoff scores that best differentiated patients likely to survive beyond three months included the Revised Tokuhashi score=10, Tomita score=four, Modified Bauer score=three, and NESMS=one. CONCLUSION: We found comparable efficacy among the models in predicting survival at clinically relevant time points. Cutoff values provided herein may assist surgeons and patients when deciding whether to pursue surgery for spinal metastatic disease. LEVEL OF EVIDENCE: 4.

Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers.

This article reports nonlinear optical measurements that quantify, for the first time directly and without labels, how many Mg(2+) cations are bound to DNA 21-mers covalently linked to fused silica/water interfaces maintained at pH 7 and 10 mM NaCl, and what the thermodynamics are of these interactions. The overall interaction of Mg(2+) with adenine, thymine, guanine, and cytosine is found to involve -10.0 ± 0.3, -11.2 ± 0.3, -14.0 ± 0.4, and -14.9 ± 0.4 kJ/mol, and nonspecific interactions with the phosphate and sugar backbone are found to contribute -21.0 ± 0.6 kJ/mol for each Mg(2+) ion bound. The specific and nonspecific contributions to the interaction energy of Mg(2+) with oligonucleotide single strands is found to be additive, which suggests that within the uncertainty of these surface-specific experiments, the Mg(2+) ions are evenly distributed over the oligomers and not isolated to the most strongly binding nucleobase. The nucleobases adenine and thymine are found to bind only three Mg(2+) ions per 21-mer oligonucleotide, while the bases cytosine and guanine are found to bind eleven Mg(2+) ions per 21-mer oligonucleotide.

Tuning Electrospun Substrate Stiffness for the Fabrication of a Biomimetic Amniotic Membrane Substitute for Corneal Healing.

Corneal blindness is the fourth most common cause of vision impairment worldwide with a high incidence in global south countries. A recently developed surgical technique for treating corneal blindness is simple limbal epithelial transplantation (SLET), which uses small pieces of healthy limbal tissue (limbal explants) delivered to the damaged eye using the human amniotic membrane (AM) as a carrier. SLET relies on the use of tissue banks for the AM that reduces the availability of the technique. Replacing the AM with a synthetic membrane is key to making SLET more accessible to those who need it. Previous research has demonstrated the suitability of electrospun poly(lactide-co-glycolide) (PLGA) scaffolds as AM substitutes, and here, we report how these membranes can be tailored to mimic fundamental AM mechanical properties. To modify the stiffness of PLGA electrospun membranes, we explored different electrospinning solvent systems (1,1,1,3,3,3,-hexafluoroisopropanol (HFIP), dichloromethane (DCM), chloroform, and N,N-dimethylformamide (DMF)) and the use of plasticizers (PEG400 and glycerol). PEG400 was found to reduce stiffness from 60 MPa to around 4 MPa, approaching the values shown by the native AM. The biocompatibility of membranes with and without PEG400 was found to be comparable, and cell outgrowth from rabbit/porcine explants was successfully observed on the materials after 3 weeks. This research underpins the manufacture of next-generation fibrous biomimetic membranes that will ultimately be used as amniotic membrane substitutes for biomedical applications including SLET.

Vector Beam Polarization State Spectrum Analyzer.

We present a proof of concept for a vector beam polarization state spectrum analyzer based on the combination of a polarization diffraction grating (PDG) and an encoded harmonic q-plate grating (QPG). As a result, a two-dimensional polarization diffraction grating is formed that generates six different q-plate channels with topological charges from -3 to +3 in the horizontal direction, and each is split in the vertical direction into the six polarization channels at the cardinal points of the corresponding higher-order Poincaré sphere. Consequently, 36 different channels are generated in parallel. This special polarization diffractive element is experimentally demonstrated using a single phase-only spatial light modulator in a reflective optical architecture. Finally, we show that this system can be used as a vector beam polarization state spectrum analyzer, where both the topological charge and the state of polarization of an input vector beam can be simultaneously determined in a single experiment. We expect that these results would be useful for applications in optical communications.

Y(III) interactions with guanine oligonucleotides covalently attached to aqueous/solid interfaces.

The binding of Y(III) ions to surface-immobilized single-stranded 20-mers of guanine was studied using the Eisenthal χ((3)) technique and AFM. The free energy of binding for Y(III) to the G(20) sequence was found to be -39.5(8) kJ/mol. Furthermore, yttrium binds much more strongly to surface-immobilized oligonucleotides than the divalent metals previously reported. At maximum surface coverage, Y(III) ion densities range between one to three ions bound per strand. Comparatively, Mg(II) binds to the G(20)-functionalized interface in much higher ion densities. This result may be explained, in part, by the larger hydration sphere radius of Y(III) compared to that of Mg(II). The ion loading and binding free energy results, in conjunction with other surface and bulk aqueous phase studies, suggest that a fully hydrated +2 or +3 yttrium ion binds to the oligonucleotides through an outer-sphere mechanism. Tapping mode AFM results indicate that oligonucleotide height does not appreciably decrease following Y(III) binding. These results, together with the low ion densities for Y(III) ions, indicate that Y(III) strand loading may not significantly decrease the intrastrand Coulombic repulsions in order to cause a significant decrease in oligomer height.

Importance of length and sequence order on magnesium binding to surface-bound oligonucleotides studied by second harmonic generation and atomic force microscopy.

The binding of magnesium ions to surface-bound single-stranded oligonucleotides was studied under aqueous conditions using second harmonic generation (SHG) and atomic force microscopy (AFM). The effect of strand length on the number of Mg(II) ions bound and their free binding energy was examined for 5-, 10-, 15-, and 20-mers of adenine and guanine at pH 7, 298 K, and 10 mM NaCl. The binding free energies for adenine and guanine sequences were calculated to be -32.1(4) and -35.6(2) kJ/mol, respectively, and invariant with strand length. Furthermore, the ion density for adenine oligonucleotides did not change as strand length increased, with an average value of 2(1) ions/strand. In sharp contrast, guanine oligonucleotides displayed a linear relationship between strand length and ion density, suggesting that cooperativity is important. This data gives predictive capabilities for mixed strands of various lengths, which we exploit for 20-mers of adenines and guanines. In addition, the role sequence order plays in strands of hetero-oligonucleotides was examined for 5'-A(10)G(10)-3', 5'-(AG)(10)-3', and 5'-G(10)A(10)-3' (here the -3' end is chemically modified to bind to the surface). Although the free energy of binding is the same for these three strands (averaged to be -33.3(4) kJ/mol), the total ion density increases when several guanine residues are close to the 3' end (and thus close to the solid support substrate). To further understand these results, we analyzed the height profiles of the functionalized surfaces with tapping-mode atomic force microscopy (AFM). When comparing the average surface height profiles of the oligonucleotide surfaces pre- and post- Mg(II) binding, a positive correlation was found between ion density and the subsequent height decrease following Mg(II) binding, which we attribute to reductions in Coulomb repulsion and strand collapse once a critical number of Mg(II) ions are bound to the strand.

Divalent metal cation speciation and binding to surface-bound oligonucleotide single strands studied by second harmonic generation.

The binding of Sr(II), Ca(II), Mg(II), Ba(II), Mn(II), Zn(II), and Cd(II) to silica/water interfaces functionalized with A(15)T(6) oligonucleotides was quantified at pH 7 and 10 mM NaCl using the Eisenthal χ((3)) technique. The binding free energies range from -31.1(6) kJ/mol for Ba(II) to -33.8(4) kJ/mol for Ca(II). The ion densities were found to range from 2(1) ions/strand for Zn(II) to 11(1) ions/strand for Cd(II). Additionally, we quantified Mg(II) binding in the presence of varying background electrolyte concentrations which showed that the binding free energies changed in a linear fashion from -39.3(8) to -27(1) kJ/mol over the electrolyte concentration range of 1-80 mM, respectively. An adsorption free energy versus interfacial potential analysis allowed us to elucidate the speciation of the bound Mg(II) ions and to identify three possible binding pathways. Our findings suggest that Mg(II) binds as a fully hydrated divalent cation, most likely displacing DNA-bound Na ions. These measurements will serve as a benchmark for computer simulations of divalent metal cation/DNA interactions for geochemical and biosensing applications.