Novel enzyme-linked aptamer-antibody sandwich assay and hybrid lateral flow strip for SARS-CoV-2 detection.

We have successfully generated oligonucleotide aptamers (Apts) and monoclonal antibodies (mAbs) targeting the recombinant nucleocapsid (N) protein of SARS-CoV-2. Apts were obtained through seven rounds of systematic evolution of ligands by exponential enrichment (SELEX), while mAbs were derived from the 6F6E11 hybridoma cell line. Leveraging these Apts and mAbs, we have successfully devised two innovative and remarkably sensitive detection techniques for the rapid identification of SARS-CoV-2 N protein in nasopharyngeal samples: the enzyme-linked aptamer-antibody sandwich assay (ELAAA) and the hybrid lateral flow strip (hybrid-LFS). ELAAA exhibited an impressive detection limit of 0.1 ng/mL, while hybrid-LFS offered a detection range of 0.1 - 0.5 ng/mL. In the evaluation using ten nasopharyngeal samples spiked with known N protein concentrations, ELAAA demonstrated an average recovery rate of 92%. Additionally, during the assessment of five nasopharyngeal samples from infected individuals and ten samples from healthy volunteers, hybrid-LFS displayed excellent sensitivity and specificity. Our study introduces a novel and efficient on-site approach for SARS-CoV-2 detection in nasopharyngeal samples. The reliable hybrid Apt-mAb strategy not only advances virus diagnostic methods but also holds promise in combating the spread of related diseases.

The influence of large-diameter multifocal contact lens on ocular surface, visual quality, and visual function for presbyopic adults with dry eye syndromes.

This study investigated the influence of large-diameter multifocal contact lenses on the ocular surface, visual quality, and visual function for presbyopic adults with dry eye syndromes. The study enrolled 40-55-year-old adults with presbyopia and dry eye syndromes (DES). The subjects were randomly assigned to three groups wearing different designs of contact lenses (Proclear, SMR, and Optimum) for 6-8 h a day for two weeks. Ocular surface health, tear quality, visual quality, and visual function were measured before and after lens wear. No significant difference was observed across all three groups for the amount of conjunctival redness, blink frequency (lens on), and stereopsis vision before and after wearing. Although there seemed to be a significant declining trend for corneal staining and limbal redness, non-invasive tear break-up time (TBUT), and lipid layer thickness while lens wear, the measured values were all within the normal range. Vice-versa after lens removal, results also showed significant improvement on lipid layer thickness, blink frequency (lens off), and contact TBUT. A significant improvement was observed in the modulation transfer function (MTF) of the total area ratio after wearing contact lenses. In contrast, the MTF of the high-order aberration area ratio resulting from lens wear was lower than that of the baseline measurement. There are also significant improvements observed for SMR and Optimum regarding near visual acuity, near point of accommodation, and the subjective questionnaire (OSDI and VBP) scores. Although it is difficult to avoid a specific negative impact on the ocular surface and tear film, visual function and visual quality can still be positively improved, especially shown on larger diameter and distance-center designed multifocal contact lenses.

Investigation of treatment satisfaction and health-related quality of life after add-on to metformin-based therapy in patients with type 2 diabetes.

Background: The complexity of oral antidiabetic drug (OAD) regimens affects the quality of life (QOL) and treatment satisfaction. However, data on the QOL of patients with type 2 diabetes mellitus (T2DM) receiving metformin-based OAD treatment in Asia are limited. Therefore, this study aimed to evaluate the QOL and treatment satisfaction and explore the influencing factors and their correlations among patients with T2DM receiving metformin-based OADs. Methods: This was a cross-sectional study conducted at the Outpatient Department of Metabolism and Endocrinology at a medical center in Taiwan. Data were collected using the Audit of Diabetes-Dependent Quality of Life (ADDQoL) and the Chinese version of the Satisfaction with Oral Anti-Diabetic Agent Scale (C-SOADAS) questionnaires from patients with T2DM using metformin. The outcomes were analyzed by group and stratified based on the use of two, three, and more than three OADs. The level of agreement between the questionnaires was analyzed using Spearman's rank correlation coefficient. Results: A total of 153 patients with T2DM using metformin were included in this study. The average weighted impact score in the ADDQoL was -2.11, with no significant differences between the three groups. The C-SOADAS score showed a significant difference between the groups using two, three, and more than three OADs (21.42 [1.98] vs. 20.43 [2.09] vs. 19.00 [2.24], p < 0.0001). The ADDQoL and C-SOADAS scores showed low correlations between patients' QOL and treatment satisfaction. However, the impact of diabetes on specific aspects of life was negatively correlated with the total C-SOADAS scores. Conclusion: In Taiwan, a significantly greater effect on QOL was observed among patients with fewer OAD classes and higher treatment satisfaction. This study provides local evidence from self-reporting outcomes of patients with T2DM. Further studies focusing on different populations and treatment regimens for QOL are needed.

Harnessing polyhydroxylated pyrrolidines as a stabilizer of acid alpha-glucosidase (GAA) to enhance the efficacy of enzyme replacement therapy in Pompe disease.

To discover small molecules as acid alpha-glucosidase (GAA) stabilizers for potential benefits of the exogenous enzyme treatment toward Pompe disease cells, we started from the initial screening of the unique chemical space, consisting of sixteen stereoisomers of 2-aminomethyl polyhydroxylated pyrrolidines (ADMDPs) to find out two primary stabilizers 17 and 18. Further external or internal structural modifications of 17 and 18 were performed to increase structural diversity, followed by the protein thermal shift study to evaluate the GAA stabilizing ability. Fortunately, pyrrolidine 21, possessing an l-arabino-typed configuration pattern, was identified as a specific potent rh-GAA stabilizer, enabling the suppression of rh-GAA protein denaturation. In a cell-based Pompe model, co-administration of 21 with rh-GAA protein significantly improved enzymatic activity (up to 5-fold) compared to administration of enzyme alone. Potentially, pyrrolidine 21 enables the direct increase of ERT (enzyme replacement therapy) efficacy in cellulo and in vivo.

Follistatin-like protein 1 functions as a potential target of gene therapy in proliferative diabetic retinopathy.

The degree of retinal fibrosis increased in proliferative diabetic retinopathy (PDR) patients after administration of anti-Vascular endothelial growth factor (VEGF) injections. Previous studies showed that the balance between connective tissue growth factor (CTGF) and VEGF plays an important role. Therefore, in a high-glucose state, an anti-VEGF and CTGFshRNA dual-target model was used to simulate clinical dual-target treatment in PDR patients, and RNA sequencing (RNA-Seq) technology was used for whole transcriptome sequencing. A hypoxia model was constructed to verify the sequencing results at the cellular level, and the vitreous humor and proliferative membranes were collected from patients for verification. All sequencing results included Follistatin-like protein 1 (FSTL1) and extracellular matrix (ECM) receptor pathway, indicated that anti-VEGF therapy may upregulate FSTL1 expression, while dual-target treatment downregulated FSTL1. Thus, we further studied the function of FSTL1 on the expression of VEGF and ECM factors by both overexpressing and silencing FSTL1. In conclusion, our results suggested that FSTL1 may be involved in the pathogenesis of PDR and is related to fibrosis caused by the anti-VEGF treatment, thus providing a potential target for gene therapy in PDR.

Design, synthesis and the structure-activity relationship of agonists targeting on the ALDH2 catalytic tunnel.

ALDH2, a key enzyme in the alcohol metabolism process, detoxifies several kinds of toxic small molecular aldehydes, which induce severe organ damages. The development of novel Alda-1 type ALDH2 activators was mostly relied on HTS but not rational design so far. To clarify the structure-activity relationship (SAR) of the skeleton of Alda-1 analogs by synthesis of the least number of analogs, we prepared 31 Alda-1 analogs and 3 isoflavone derivatives and evaluated for their ALDH2-activating activity. Among these, the ALDH2-activating activity of mono-halogen-substituted (Cl and Br) N-piperonylbenzamides 3b and 3 k, and non-aromatic amides 8a-8c, were 1.5-2.1 folds higher than that of Alda-1 at 20 µM. The relationship between binding affinity in computer aided molecular docking model and the ALDH2-activating activity assays were clarified as follows: for Alda-1 analogs, with the formation of halogen bonds, the enzyme-activating activity was found to follow a specific regression curve within the range between -5 kcal/mol and -4 kcal/mol. For isoflavone derivatives, the basic moiety on the B ring enhance the activating activity. These results provide a new direction of utilizing computer-aided modeling to design novel ALDH2 agonists in the future.

Antidepressant-resistant depression is characterized by reduced short- and long-interval cortical inhibition.

BACKGROUND: Major depressive disorder (MDD) is highly heterogeneous and can be classified as treatment-resistant depression (TRD) or antidepressant-responsive depression (non-TRD) based on patients' responses to antidepressant treatment. Methods for distinguishing between TRD and non-TRD are critical clinical concerns. Deficits of cortical inhibition (CI) have been reported to play an influential role in the pathophysiology of MDD. Whether TRD patients' CI is more impaired than that of non-TRD patients remains unclear. METHODS: Paired-pulse transcranial magnetic stimulation (ppTMS) was used to measure cortical inhibitory function including GABAA- and GABAB-receptor-related CI and cortical excitatory function including glutamate-receptor-related intracortical facilitation (ICF). We recruited 36 healthy controls (HC) and 36 patients with MDD (non-TRD, n = 16; TRD, n = 20). All participants received evaluations for depression severity and ppTMS examinations. Non-TRD patients received an additional ppTMS examination after 3 months of treatment with the SSRI escitalopram. RESULTS: Patients with TRD exhibited reduced short-interval intracortical inhibition (SICI) and long-interval intracortical inhibition (LICI), as shown by abnormally higher estimates, than those with non-TRD or HC (F = 11.030, p < 0.001; F = 10.309, p < 0.001, respectively). After an adequate trial of escitalopram treatment, the LICI of non-TRD reduced significantly (t = - 3.628, p < 0.001), whereas the ICF remained lower than that of HC and showed no difference from pretreatment non-TRD. CONCLUSIONS: TRD was characterized by relatively reduced CI, including both GABAA- and GABAB-receptor-mediated neurons while non-TRD preserved partial CI. In non-TRD, SSRIs may mainly modulate GABAB-receptor-related LICI. Our findings revealed distinguishable features of CI in antidepressant-resistant and responsive major depression.

Incomplete fluid-air exchange technique for idiopathic macular hole surgery.

AIM: To explore an improved procedure involving incomplete fluid-air exchange for idiopathic macular hole (IMH), and the closure rate, visual function, and the visual field of macular holes (MHs) were evaluated. METHODS: This prospective randomized controlled study, included 40 eyes of 40 patients with IMH who were treated with pars plana vitrectomy and peeling of the internal limiting membrane. They were grouped by random digital table. Twenty-one eyes underwent incomplete fluid-air exchange (IFA) and 19 eyes underwent traditional complete fluid-air exchange (CFA) as the control group. Outcomes included best-corrected visual acuity (BCVA), intraocular pressure, and optical coherence tomography, light adaptive electroretinography, and visual field evaluations. RESULTS: All MHs <400 µm were successfully closed. BCVAs before and 6mo after surgery were 0.82±0.41 logMAR and 0.28±0.17 logMAR in IFA group and 0.86±0.34 logMAR and 0.34±0.23 logMAR in CFA group, respectively. The electroretinogram analysis of patients in IFA group revealed increases in b-wave amplitudes at 1, 3, and 6mo after surgery. Additionally, patients in IFA group showed an amplitude increase of 28.6% from baseline at 6mo (P<0.05), while no obvious improvements were noted in CFA group. Although there were no statistically significant improvements in either group, the IFA group showed a slight increase in mean sensitivity (P>0.05). CONCLUSION: IFA is a reliable method that offers comparable closure rate to CFA and facilitates improvements in visual function.

Dexmedetomidine modulates neuroinflammation and improves outcome via alpha2-adrenergic receptor signaling after rat spinal cord injury.

BACKGROUND: Spinal cord injury induces inflammatory responses that include the release of cytokines and the recruitment and activation of macrophages and microglia. Neuroinflammation at the lesion site contributes to secondary tissue injury and permanent locomotor dysfunction. Dexmedetomidine (DEX), a highly selective α2-adrenergic receptor agonist, is anti-inflammatory and neuroprotective in both preclinical and clinical trials. We investigated the effect of DEX on the microglial response, and histological and neurological outcomes in a rat model of cervical spinal cord injury. METHODS: Anaesthetised rats underwent unilateral (right) C5 spinal cord contusion (75 kdyne) using an impactor device. The locomotor function, injury size, and inflammatory responses were assessed. The effect of DEX was also studied in a microglial cell culture model. RESULTS: DEX significantly improved the ipsilateral upper-limb motor dysfunction (grooming and paw placement; P<0.0001 and P=0.0012), decreased the injury size (P<0.05), spared white matter (P<0.05), and reduced the number of activated macrophages (P<0.05) at the injury site 4 weeks post-SCI. In DEX-treated rats after injury, tissue RNA expression indicated a significant downregulation of pro-inflammatory markers (e.g. interleukin [IL]-1ß, tumour necrosis factor-α, interleukin (IL)-6, and CD11b) and an upregulation of anti-inflammatory and pro-resolving M2 responses (e.g. IL-4, arginase-1, and CD206) (P<0.05). In lipopolysaccharide-stimulated cultured microglia, DEX produced a similar inflammation-modulatory effect as was seen in spinal cord injury. The benefits of DEX on these outcomes were mostly reversed by an α2-adrenergic receptor antagonist. CONCLUSIONS: DEX significantly improves neurological outcomes and decreases tissue damage after spinal cord injury, which is associated with modulation of neuroinflammation and is partially mediated via α2-adrenergic receptor signaling.

Prevalence of isomeric plastomes and effectiveness of plastome super-barcodes in yews (Taxus) worldwide.

Taxus (yew) is both the most species-rich and taxonomically difficult genus in Taxaceae. To date, no study has elucidated the complexities of the plastid genome (plastome) or examined the possibility of whole plastomes as super-barcodes across yew species worldwide. In this study, we sequenced plastomes from two to three individuals for each of the 16 recognized yew species (including three potential cryptics) and Pseudotaxus chienii. Our comparative analyses uncovered several gene loss events that independently occurred in yews, resulting in a lower plastid gene number than other Taxaceous genera. In Pseudotaxus and Taxus, we found two isomeric arrangements that differ by the orientation of a 35 kb fragment flanked by "trnQ-IRs". These two arrangements exist in different ratios within each sampled individual, and intraspecific shifts in major isomeric arrangements are first reported here in Taxus. Moreover, we demonstrate that entire plastomes can be used to successfully discriminate all Taxus species with 100% support, suggesting that they are useful as super-barcodes for species identification. We also propose that accD and rrn16-rrn23 are promising special barcodes to discriminate yew species. Our newly developed Taxus plastomic sequences provide a resource for super-barcodes and conservation genetics of several endangered yews and serve as comprehensive data to improve models of plastome complexity in Taxaceae as a whole and authenticate Taxus species.

Detection of local and remote cellular damage caused by spinal cord and peripheral nerve injury using a heat shock signaling reporter system.

Spinal cord and peripheral nerve injury results in extensive damage to the locally injured cells as well as distant cells that are functionally connected to them. Both primary and secondary damage can cause a broad range of clinical abnormalities, including neuropathic pain and cognitive and memory dysfunction. However, the mechanisms underlying these abnormalities remain unclear, awaiting new methods to identify affected cells to enable examination of their molecular, cellular and physiological characteristics. Here, we report that both primary and secondary damage to cells in mouse models of spinal cord and peripheral nerve injury can be detected in vivo using a novel fluorescent reporter system based on the immediate stress response via activation of Heat Shock Factor 1. We also provide evidence for altered electrophysiological properties of reporter-positive secondarily-injured neurons. The comprehensive identification of injured, but surviving cells located both close and at distant locations from the injury site in vivo will provide a way to study their pathophysiology and possibly prevention of their further deterioration.

Gene expression changes in honey bees induced by sublethal imidacloprid exposure during the larval stage.

Honey bee larvae exposed to sublethal doses of imidacloprid show behavioural abnormalities as adult insects. Previous studies have demonstrated that this phenomenon originates from abnormal neural development in response to imidacloprid exposure. Here, we further investigated the global gene expression changes in the heads of newly emerged adults and observed that 578 genes showed more than 2-fold changes in gene expression after imidacloprid exposure. This information might aid in understanding the effects of pesticides on the health of pollinators. For example, the genes encoding major royal jelly proteins (MRJPs), a group of multifunctional proteins with significant roles in the sustainable development of bee colonies, were strongly downregulated. These downregulation patterns were further confirmed through analyses using quantitative reverse transcription-polymerase chain reaction on the heads of 6-day-old nurse bees. To our knowledge, this study is the first to demonstrate that sublethal doses of imidacloprid affect mrjp expression and likely weaken bee colonies.

Detection of vulnerable neurons damaged by environmental insults in utero.

Development of prognostic biomarkers for the detection of prenatally damaged neurons before manifestations of postnatal disorders is an essential step for prevention and treatment of susceptible individuals. We have developed a versatile fluorescence reporter system in mice enabling detection of Heat Shock Factor 1 activation in response to prenatal cellular damage caused by exposure to various harmful chemical or physical agents. Using an intrautero electroporation-mediated reporter assay and transgenic reporter mice, we are able to identify neurons that survive prenatal exposure to harmful agents but remain vulnerable in postnatal life. This system may provide a powerful tool for exploring the pathogenesis and treatment of multiple disorders caused by exposure to environmental stress before symptoms become manifested, exacerbated, and/or irreversible.

Chimeric peptide containing both B and T cells epitope of tumor-associated antigen L6 enhances anti-tumor effects in HLA-A2 transgenic mice.

Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. In this report, we identified a new B cell epitope of tumor-associated antigen L6 (TAL6) that could induce antibody-dependent cellular cytotoxicity (ADCC) in vivo. We incorporated the B cell epitope with a cytotoxic T lymphocyte (CTL) and a helper T (Th) epitope to form a chimeric long peptide. We formulated the chimeric peptide with different adjuvants to immunize HLA-A2 transgenic mice and evaluate their immunogenicity. The chimeric peptide formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC and CTL responses. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells. Moreover, we observed that immunization with the chimeric peptide inhibited cancer cell migration in vitro and metastasis in vivo. These data suggest that a chimeric peptide containing both B and T cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy.

Gemcitabine enhances antitumor efficacy of recombinant lipoimmunogen-based immunotherapy.

Although immunotherapy is an attractive approach for cancer treatment, increasing evidence has shown that the combination of immunotherapy with other treatment modalities may improve the outcome of advanced malignancy. We combined the anticancer drug gemcitabine (Gem) with recombinant lipoprotein-based immunotherapy (rlipo-E7m/CpG) to treat advanced cancer. Mice bearing huge solid tumors (≧ 12 mm in diameter) or orthotopic cervical cancer were treated with a therapeutic regimen consisting of rlipo-E7m/CpG and Gem. In addition, tumor-infiltrating immune cells were quantified by flow cytometry following the chemotherapy and/or immunotherapy. We observed the eradication of huge tumors following the administration of Gem on days 21, 24, and 27 or following rlipo-E7m/CpG therapy on day 30 post-tumor implantation. The combination therapy substantially reduced the number of immunosuppressive cells (CD11b+Gr-1+, CD11b+F4/80+, and CD4+CD25+FOXP3+) and increased the number of tumor-infiltrating antigen-specific CD8+ T cells compared to Gem or rlipo-E7m/CpG monotherapy. Interestingly, the administration of Gem and rlipo-E7m/CpG reduced the quantity of programmed cell death protein 1 (PD-1)-expressing antigen-specific cytotoxic T lymphocytes (CTLs) in the regressing tumors. These findings demonstrated that Gem enhances the eradication of huge tumors by inhibiting a broad range of immunosuppressive cells when combined with immunotherapy. Based on the promising results from this animal study, Gem chemotherapy combined with recombinant lipoimmunogen-based immunotherapy represents a feasible approach for cancer therapy.

Combined online and offline adaptive radiation therapy: a dosimetric feasibility study.

PURPOSE: The purpose of this work is to explore a new adaptive radiation therapy (ART) strategy, combined "online and offline" ART, that can fully account for interfraction variations similar to the existing online ART but with substantially reduced online effort. METHODS AND MATERIALS: The concept for the combined ART is to perform online ART only for the fractions with obvious interfraction variations and to deliver the ART plan for that online fraction as well as the subsequent fractions until the next online fraction needs to be adapted. To demonstrate the idea, the daily computed tomographic (CT) data acquired during image guided radiation therapy (IGRT) with an in-room CT (CTVision, Siemens Healthcare, Amarillo, TX) for 6 representative patients (including 2 prostate, 1 head-and-neck, and 1 pancreatic cancer, 1 adrenal carcinoma, and 1 craniopharyngioma patients) were analyzed. Three types of plans were generated based on the following selected daily CTs: (1) IGRT repositioning plan, generated by applying the repositioning shifts to the original plan (representing the current IGRT practice); (2) Re-Opt plan, generated with full-scope optimization; and (3) ART plan, either online ART plan generated with an online ART tool (RealArt, Prowess Inc, Concord, CA) or offline ART plan generated with shifts from the online ART plan. Various dose-volume parameters were compared with measure dosimetric benefits of the ART plans based on daily dose distributions and the cumulative dose maps obtained with deformable image registration. RESULTS: In general, for all the cases studied, the ART (with 3-5 online ART) and Re-Opt plans provide comparable plan quality and offer significantly better target coverage and normal tissue sparing when compared with the repositioning plans. This improvement is statistically significant. CONCLUSIONS: The combined online and offline ART is dosimetrically equivalent to the online ART but with substantially reduced online effort, and enables immediate delivery of the adaptive plan when an obvious anatomic change is observed.

Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury.

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV-V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.

A planning and delivery study of a rotational IMRT technique with burst delivery.

PURPOSE: A novel rotational IMRT (rIMRT) technique using burst delivery (continuous gantry rotation with beam off during MLC repositioning) is investigated. The authors evaluate the plan quality and delivery efficiency and accuracy of this dynamic technique with a conventional flat 6 MV photon beam. METHODS: Burst-delivery rIMRT was implemented in a planning system and delivered with a 160-MLC linac. Ten rIMRT plans were generated for five anonymized patient cases encompassing head and neck, brain, prostate, and prone breast. All plans were analyzed retrospectively and not used for treatment. Among the varied plan parameters were the number of optimization points, number of arcs, gantry speed, and gantry angle range (alpha) over which the beam is turned on at each optimization point. Combined rotational/step-and-shoot rIMRT plans were also created by superimposing multiple-segment static fields at several optimization points. The rIMRT trial plans were compared with each other and with plans generated using helical tomotherapy and VMAT. Burst-mode rotational IMRT plans were delivered and verified using a diode array, ionization chambers, thermoluminescent dosimeters, and film. RESULTS: Burst-mode rIMRT can achieve plan quality comparable to helical tomotherapy, while the former may lead to slightly better OAR sparing for certain cases and the latter generally achieves slightly lower hot spots. Few instances were found in which increasing the number of optimization points above 36, or superimposing step-and-shoot IMRT segments, led to statistically significant improvements in OAR sparing. Using an additional rIMRT partial arc yielded substantial OAR dose improvements for the brain case. Measured doses from the rIMRT plan delivery were within 4% of the plan calculation in low dose gradient regions. Delivery time range was 228-375 s for single-arc rIMRT 200-cGy prescription with a 300 MU/min dose rate, comparable to tomotherapy and VMAT. CONCLUSIONS: Rotational IMRT with burst delivery, whether combined with static fields or not, yields clinically acceptable and deliverable treatment plans.

Schwann cell proliferation and macrophage infiltration are evident at day 14 after painful cervical nerve root compression in the rat.

Although it is known that different types of nerve root insults can produce radicular pain, it is not known whether the neuronal and Schwann cell pathologies in the nerve root vary between inflammation-induced nerve root injury and traumatic compression. This study examined the extent of Wallerian degeneration and associated cellular repair processes in the nerve root in the context of mechanical hyperalgesia resulting from different modes of painful nerve root injury. The C7 dorsal nerve root underwent a transient 10 gram-force compression (10 g), inflammation-induced irritation by chromic gut exposure (Cg), or a combination of those stimuli (10 g+Cg). Fourteen days after injury when hyperalgesia remained, immunohistochemical analysis revealed upregulation of substance P, robust macrophage infiltration, myelin degeneration and debris removal, and a significant increase in the number of myelinating Schwann cells (Krox20-positive) in the compressed roots (10 g, 10 g+Cg). Cg alone also produced hyperalgesia, despite being associated with intact myelin. Unilateral exposure to chromic material induced bilateral increases in macrophages and Krox20-positive Schwann cells in the nerve roots, and substance P expression in the dorsal root ganglion (DRG) neurons. Results suggest that despite similar sensitivity, the extent of infiltrating macrophages and repopulated Schwann cells varies for pain from mechanical and/or chemical nerve root injury. Although these different cellular mechanisms may explain pain, they may also only reflect varying injury etiologies.

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.