Codelivery of TGFß and Cox2 siRNA inhibits HCC by promoting T-cell penetration into the tumor and improves response to Immune Checkpoint Inhibitors.

Upregulation of TGFß and Cox2 in the tumor microenvironment results in blockade of T-cell penetration into the tumor. Without access to tumor antigens, the T-cell response will not benefit from administration of the immune checkpoint antibodies. We created an intravenous polypeptide nanoparticle that can deliver two siRNAs (silencing TGFß and Cox2). Systemic administration in mice, bearing a syngeneic orthotopic hepatocellular carcinoma (HCC), delivers the siRNAs to various cells in the liver, and significantly reduces the tumor. At 2 mg/kg (BIW) the nanoparticle demonstrated a single agent action and induced tumor growth inhibition to undetectable levels after five doses. Reducing the siRNAs to 1mg/kg BIW demonstrated greater inhibition in the presence of PD-L1 mAbs. After only three doses BIW, we could still recover a smaller tumor and, in tumor sections, showed an increase in penetration of CD4+ and CD8+ T-cells deeper into the remaining tumor that was not evident in animals treated with non-silencing siRNA. The combination of TGFß and Cox2 siRNA co-administered in a polypeptide nanoparticle can act as a novel therapeutic alone against HCC and may augment the activity of the immune checkpoint antibodies. Silencing TGFß and Cox2 converts an immune excluded (cold) tumor into a T-cell inflamed (hot) tumor.

Synthesis of Nucleotides Bearing the 2'-O-Trifluoromethyl Group and Their Application in RNA Analogs Preparation.

The integration of fluorine atoms into biologically active organic compounds has proved to be a vital technique in small molecule drugs. This technique can substantially enhance crucial properties, including metabolic stability, lipophilicity, and bioavailability, often with a mere addition of a single fluorine atom or a trifluoromethyl group. Over the past few decades, this concept has also been applied in nucleic acid chemistry. A commonly employed 2'-OH substitution is the introduction of a 2'-deoxy-2'-fluoro (2'-F) group. The strong electronegativity of fluorine prompts the modified siRNA to readily adopt a C3'-endo conformation, resulting in significant advantages in terms of binding affinity. To enrich the toolbox of chemical modification of oligonucleotides, the replacement of the 2'-OH with the 2'-O-trifluoromethyl group has been developed in RNA analog synthesis. Oligodeoxynucleotides containing the 2'-O-trifluoromethyl group can greatly increase the thermal stability of DNA/RNA duplexes depending on the position and amount of the modification. Moreover, 2'-O-trifluoromethylated oligodeoxynucleotide also exhibited a slightly higher resistance to snake venom phosphodiesterase than the unmodified oligodeoxynucleotide. The 2'-O-trifluoromethylated oligonucleotides can emerge as a label to study RNA structure and function as well, or to develop DNA/RNA-based diagnostics. Hence, it is necessary to report an effective method for the synthesis, deprotection, purification, and characterization of oligonucleotides bearing a 2'-O-trifluoromethyl group. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 6-N-benzoyl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl adenosine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 2: Preparation of 4-N-acetyl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl cytidine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 3: Preparation of 2-N-isobutyryl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl guanine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 4: Preparation of 5'-O-dimethoxytrityl-2'-O-2-trifluoromethyl uridine 3'-(2-cyanoethyl N,N-diisopropyl) phosphoramidite Basic Protocol 5: Solid-phase synthesis of 2'-O-trifluoromethylated RNA analogs Basic Protocol 6: Deprotection and purification of 2'-O-trifluoromethyl-RNAs.

Rising use of diagnostic imaging in Australia: An analysis of Medicare-funded radiology services between 2000 and 2021.

INTRODUCTION: The use of diagnostic imaging services is increasing worldwide. This has important impacts on healthcare resource allocation and potential risks to the population. This study aimed to quantify trends in medical imaging in Australia over the past two decades. METHODS: Data were extracted from the Australian Medicare Benefits Schedule (MBS) between 2000 and 2021. Simple linear regression analyses were performed to assess changes in absolute utilisation and utilisation rate per 100,000 population of total imaging services as well as by each imaging modality. Logistic regression analysis was performed to assess changes in total imaging services as a proportion of total Medicare services over time. Chi-squared test was used to assess for change in modality composition of imaging services. RESULTS: There were 436,255,500 imaging studies performed between 2000 and 2021. The absolute utilisation of total imaging services increased annually by an average of 864,404 (95% CI: 808,235-920,573, p < 0.001). For each consecutive year, the proportion of total Medicare services attributed to total imaging services increased by 0.01% (95% CI: 0.01-0.01, p < 0.01). There was also a statistically significant increase in the utilisation rates of imaging services per 100,000 population for each imaging modality. The number of imaging services per radiologist increased on average by 74 (95% CI: 26-122, p < 0.05) annually. CONCLUSION: The utilisation of diagnostic imaging services has increased in Australia between 2000 and 2021, outpacing the population growth, total healthcare services, and the radiologist workforce.

A Convenient Method for the Synthesis of 2'-O-Cyanoethylated Nucleotides and Their Application in the Solid-Phase Synthesis of Related RNA Analogs.

Although small interfering RNA (siRNA) is a key player among gene inhibition therapeutics, there are many obstacles to the development of siRNA drugs due to inherent properties of oligonucleotides, including the unsatisfactory stability of unmodified siRNA, poor pharmacokinetic distribution, and the toxicity induced by off-target effects. To maximize treatment potency, chemical modification of siRNA has undoubtedly been the most successful strategy by far. Widely applied modifications include phosphorothioate linkages, 2'-O-methyl modifications, and 2'-fluoro modifications, among others. To extend the family of chemical modifications for oligonucleotides, 2'-O-cyanoethylated RNA analogs were developed through the replacement of the 2'-hydroxyl group with a 2'-O-cyanoethyl group (-OCH2 CH2 CN). This modification can provide several advantages over unmodified RNA, such as increased stability, improved binding affinity to complementary DNA or RNA strands, and resistance to degradation by cellular nucleases. The 2'-O-cyanoethyl-modified RNAs not only are applied in RNA silencing machinery but also act as research tools for studying RNA structure and function or for developing RNA-based diagnostics. Therefore, the efficient synthesis, deprotection, purification, and characterization of 2'-O-cyanoethylated RNAs deserves more attention. This protocol describes the chemical synthesis of 2'-O-cyanoethylated nucleotides and the solid-phase synthesis, deprotection, and purification of 2'-O-cyanoethylated RNAs. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 6-N-dimethylformamidyl-5'-O-dimethoxytrityl-2'-O-cyanoethyl adenosine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 2: Preparation of 4-N-acetyl-5'-O-dimethoxytrityl-2'-O-cyanoethyl cytidine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 3: Preparation of 2-N-dimethylformamidyl-5'-O-dimethoxytrityl-2'-O-cyanoethyl guanine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 4: Preparation of 5'-O-dimethoxytrityl-2'-O-2-cyanoethyl uridine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 5: Solid-phase synthesis of 2'-O-cyanoethylated RNA analogs Basic Protocol 6: Deprotection and purification of synthesized 2'-O-cyanoethyl-RNAs.

Safety and Efficacy of TGF-ß1/COX-2 Silencing Therapeutic in Adults With Cutaneous Squamous Cell Carcinoma In Situ.

This single-center, open label, dose escalation cohort study evaluated the safety and efficacy of various doses of intralesional injections of TGF-β1/COX-2 combined with histidine-lysine polypeptide (siRNA/HKP) nanoparticle silencing therapeutic in patients with cutaneous in situ squamous cell carcinoma. Twenty-five patients (mean age: 67, SD: 10 years; 52% men) with cutaneous in situ squamous cell carcinoma participated. TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic was injected weekly for up to 6 weeks based on the following dosing cohorts: 10 μg/treatment, 20 μg/treatment, 30 μg/treatment, 60 μg/treatment, and 120 μg/treatment. The primary endpoint was the proportion of subjects with complete histological clearance. Also evaluated were the incidence/severity of treatment emergent adverse events and serious adverse events and incidence/severity of Local Skin Response. Twenty-five subjects received the TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic; 19 (76%) achieved histological clearance. In the 30 μg/treatment group and 60 μg/treatment group, percent cleared was 80% and 100%, respectively. Five subjects had 7 adverse events. There were no severe or serious adverse events; none led to treatment discontinuation, study interruption, or were related to the investigational product. Local skin response was none to minimal in most subjects, with improvement observed in the 10 μg/treatment, 20 μg/treatment, 30 μg/treatment, and 60 μg/treatment cohorts. Intralesional TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic injections appear to be noninvasive, safe, and efficacious in treating cutaneous in situ squamous cell carcinoma. The recommended doses for future study of the investigational product are 30 μg/treatment and 60 μg/treatment. J Drugs Dermatol. 2022;21(5):472-477. doi:10.36849/JDD.6384.

Performance of BEAMS Risk of Death Score for Mortality Prediction in Australian and New Zealand Burns Patients.

The BEAMS (Burns Evaluation and Mortality Study) risk of death score was developed in 2013 as a mortality prediction tool for burns patients admitted to an ICU (intensive care unit) in Australia and New Zealand. While it previously performed well, identifying high-risk groups and allowing benchmarking, over time such scores may lose calibration or be superseded by improved scoring systems. Our aim was to assess the performance of the BEAMS score in a modern cohort of burns patient. Data were sourced from the Burns Registry of Australia and New Zealand (BRANZ) and the Australia New Zealand Intensive Care Society Centre for Outcome and Resource Evaluation (ANZICS CORE) databases. Data were linked using probabilistic methodology. BEAMS risk of death scores was calculated for all adult patients. Between 2009 and 2019, there were 2075 patients admitted to an Australian or New Zealand ICU with a burn-related injury. Advanced age, female gender, higher %TBSA burns, and inhalation injury were all associated with increased rate of mortality (P < .05). Overall hospital mortality was 9.4% (n = 195). The predicted risk of death from BEAMS was 8.7% and the score had an area under the receiver operating characteristic curve of 0.934. We found the BEAMS risk of death score continues to have excellent performance in a modern cohort of adult critically ill burns patients. It remains a valid tool for mortality prediction among adult burns patients admitted to ICU across Australia and New Zealand.

Health-related quality of life in necrotising soft tissue infection survivors: perspective from an Australian tertiary referral centre.

BACKGROUND: Necrotising soft tissue infection (NSTI) is a life-threatening disease with widespread tissue destruction. Immediate and aggressive surgical debridement remains the main focus of treatment. This results in disfiguring scars, functional limitation and psychological sequelae for survivors. As mortality rate declines with improvements in care, a greater focus should be placed upon the psychological and functional outcomes of survivors. This study aims to assess the health-related quality of life (HRQoL) of patients following NSTI using the Short Form-36 (SF-36) and Derriford Appearance Scale-24 (DAS-24). METHODS: All NSTI patients admitted at our tertiary referral centre between 1 January 2013 and 31 December 2019 were invited to complete the DAS-24 and SF-36 surveys. A retrospective chart review was also performed. RESULTS: A total of 30 participants responded to the surveys. On comparison against the general Australian population, the NSTI cohort demonstrated significantly reduced physical and mental HRQoL as measured by the SF-36 (P < 0.001). Increased age was significantly associated with a reduced physical HRQoL (P = 0.002), while dysfunction with appearance as measured by the DAS-24 form correlated with both reduced physical and mental HRQoL (P = 0.020). A total of 79.3% of patients expressed concern regarding their appearance with a significantly higher level of distress at their appearance compared to a non-clinical population (P = 0.120). CONCLUSION: Despite the rarity of NSTI, this study demonstrates that this disease has a large and persistent burden for survivors, who report significantly reduced HRQoL and distress with appearance. Further research into comprehensive physical and psychosocial services for NSTI survivors is required.

Venous thromboembolism in burns patients: Are we underestimating the risk and underdosing our prophylaxis?

BACKGROUND: Burns patients exhibit all factors of Virchow's triad and are thus at high theoretical risk of venous thromboembolism (VTE). At our tertiary referral burns unit, a standard dose of low molecular weight heparin, which acts primarily by inhibiting Factor Xa, is given for thromboprophylaxis. However, the pharmacokinetics of enoxaparin are altered following a burn injury, and thus burns patients are likely underdosed on their thromboprophylaxis. The objectives of this study were to determine the incidence and risk factors for VTE among burns patients at the Victorian Adult Burns Service (VABS) and to determine the adequacy of the current enoxaparin thromboprophylaxis regimen through measurement of anti-factor Xa (AFXa) levels and comparison with established reference ranges. METHODS: This study consisted of two parts. In part 1, the Burns Registry of Australia and New Zealand (BRANZ) was reviewed for cases of VTE in burns patients admitted to the VABS from 2013 - 2018. Part 2 was a prospective study that determined peak and trough AFXa levels in patients admitted to the VABS with >10% total body surface area (TBSA) burns. RESULTS: Part 1. Totally, 1,475 patients were admitted to the VABS between 2013 - 2018. There were 20 cases of VTE (1.36%). Percent TBSA of burn (OR = 1.04, 95% CI: 1.03 - 1.06), full thickness burns (OR = 2.78, 95% CI: 1.15 - 6.73), ICU admission (OR = 15.08, 95% CI: 5.01 - 45.44), mechanical ventilation (OR = 10.62, 95% CI: 4.05 - 27.91), operative procedures (OR = 1.43, 95% CI: 1.29 - 1.59), and a longer hospital stay (OR = 1.05, 95% CI: 1.04 - 1.07) were all associated with an increased VTE risk. Part 2. A total of 20 participants with >10% TBSA burns were recruited to the prospective study. Peak anti Factor Xa (AFXa) levels were measured for all 20 participants with 15% recording an initial prophylactic peak AFXa level within reference range. Upon subsequent measurements, 50% of participants reached a prophylactic peak AFXa level. Trough AFXa levels were measured for 17 participants with no participant recording an initial or subsequent trough AFXa level at or above the prophylactic threshold. CONCLUSION: Our study demonstrates a high incidence of VTE among burns patients at the VABS, especially among the major burns patients, and a thromboprophylaxis protocol that is ineffective in achieving prophylactic levels of AFXa level. The evidence suggests a need to evaluate different dosing protocols among burns patients in order to improve AFXa levels, with the aim of decreasing incidence of VTE in high-risk patients.

Effects of a short message service (SMS) by cellular phone to improve compliance with fasting guidelines in patients undergoing elective surgery: a retrospective observational study.

BACKGROUND: Contemporary perioperative fasting guidelines aim to alleviate patient discomfort before surgery and enhance postoperative recovery whilst seeking to reduce the risk of pulmonary aspiration during anesthesia. The impact of a short message service (SMS) reminder on fasting guideline compliance is unknown. Therefore, we performed a retrospective observational study and quality improvement project aiming to quantify the extent of excessive and prolonged fasting, and then assessed the impact of a SMS reminder in reducing fasting times. METHODS: After ethics committee approval we performed a retrospective observational study investigating preoperative fasting times of adult patients undergoing elective surgery. First, we assessed whether the fasting guideline times were adhered to (Standard Care group). All patients received internationally recommended fasting guidelines in the form of a written hospital policy document. We then implemented an additional prompt via a mobile phone SMS 1 day prior to surgery containing a reminder of fasting guideline times (SMS group). The primary aims were to compare fasting times between the Standard Care group and the SMS group. RESULTS: The fasting times of 160 patients in the Standard Care group and 110 patients in the SMS group were evaluated. Adherence to the fasting guidelines for solids occurred in 14 patients (8.8%) in the Standard Care group vs. Twenty-two patients (13.6%) in the SMS group (p=0.01). Adherence to the fasting guidelines for fluids occurred in 4 patients (2.5%) in the Standard Care group vs. Ten patients (6.3%) in the SMS group (p=0.023). Patients in the Standard Care group had a longer median (inter-quartile range (IQR)) fasting time for fluids compared the SMS group [6.5 h (IQR 4.5:11) vs 3.5 h (IQR 3:8.5), p< 0.0001]. Median fasting times for solids were 11 h (IQR 7:14) in the Standard Care group and 11.5 h (IQR 7:13.5) in the SMS group (p=0.756). CONCLUSION: Adherence to internationally recommended fasting guidelines for patients undergoing elective surgery is poor. The introduction of a fasting guideline reminder via a mobile phone SMS in addition to a written hospital policy improved adherence to fasting advice and reduced the fasting times for fluids but not for solids. The use of an SMS reminder of fasting guidelines is a simple, feasible, low-cost, and effective tool in minimising excessive fasting for fluids among elective surgical patients. TRIAL REGISTRATION: ACTRN12619001232123 (Australia New Zealand Clinical Trials Registry). Registered 6th September 2019 (retrospectively registered).

A novel siRNA-gemcitabine construct as a potential therapeutic for treatment of pancreatic cancer.

The non-nucleoside analog gemcitabine has been the standard of care for treating pancreatic cancer. The drug shows good potency in pancreatic cancer cells in vitro but, due to poor bioavailability, requires administration in large doses by infusion and this systemic exposure results in significant toxicity for the patient. Genes have been identified that, when silenced by siRNA, synergize with gemcitabine treatment and offer a means of reducing the gemcitabine dosage required for efficacy. However, benefiting from the synergism between the two agents requires that the gemcitabine and siRNA penetrate the same cells. To ensure co-delivery, we incorporated gemcitabine covalently within siRNAs against targets synergistic with gemcitabine (CHK1 or RAD17). We demonstrated that specific bases within an siRNA can be replaced with gemcitabine to increase efficacy. The result is a single drug molecule that simultaneously co-delivers gemcitabine and a synergistic siRNA. The siRNA-gemcitabine constructs demonstrate a 5-30-fold improvement in potency compared with gemcitabine alone. Co-delivering a CHK1 siRNA-gemcitabine construct together with a WEE1 siRNA resulted in a 10-fold improvement in IC50 compared with gemcitabine alone. These constructs demonstrate efficacy across a wide array of pancreatic tumor cells and may represent a novel therapeutic approach for treating pancreatic cancer.

Case report: Iatrogenic brachial artery dissection with complete anterograde occlusion during elective arterial line placement.

INTRODUCTION: Brachial arterial catheters provide a more accurate reflection of central aortic arterial pressure compared to their radial counterparts. Although brachial arterial line complications are uncommon, we report a case of a rare iatrogenic brachial artery dissection with complete anterograde occlusion from elective arterial line placement. PRESENTATION OF CASE: A 41-year-old female presented for a right upper and middle lobe resection of a large neuroendocrine lung cancer. A brachial arterial line was inserted for continuous blood pressure monitoring using clinical landmarks. Six hours postoperatively the left hand was noted to be pale, cool and pulseless with complete paraesthesia. Thrombus was initially suspected on computed tomography angiography. Upon return to theatre, extensive dissection of the posterior brachial arterial wall was identified. CONCLUSION: We review our diagnostic pathway and treatment of this rare complication. Recommendations to minimise the risks of complications from brachial arterial line insertion are also overviewed. We recommend the routine utilization of ultrasound-guided technique and regular post-insertion neurovascular monitoring for the prevention and early recognition of complications from brachial artery catheter insertion.

Simultaneous silencing of TGF-ß1 and COX-2 reduces human skin hypertrophic scar through activation of fibroblast apoptosis.

Excessive skin scars due to elective operations or trauma represent a challenging clinical problem. Pathophysiology of hypertrophic scars entails a prolonged inflammatory and proliferative phase of wound healing. Over expression of TGF-ß1 and COX-2 play key regulatory roles of the aberrant fibrogenic responses and proinflammatory mediators. When we silenced TGF-ß1 and COX-2 expression simultaneously in primary human fibroblasts, a marked increase in the apoptotic cell population occurred in contrast to those only treated with either TGF-ß1 or COX-2 siRNA alone. Furthermore, using human hypertrophic scar and skin graft implant models in mice, we observed significant size reductions of the implanted tissues following intra-scar administration of TGF-ß1/COX-2 specific siRNA combination packaged with Histidine Lysine Polymer (HKP). Gene expression analyses of those treated tissues revealed silencing of the target gene along with down regulations of pro-fibrotic factors such as α-SMA, hydroxyproline acid, Collagen 1 and Collagen 3. Using TUNEL assay detection, we found that the human fibroblasts in the implanted tissues treated with the TGF-ß1/COX-2siRNAs combination exhibited significant apoptotic activity. Therefore we conclude that a synergistic effect of the TGF-ß1/COX-2siRNAs combination contributed to the size reductions of the hypertrophic scar implants, through activation of fibroblast apoptosis and re-balancing between scar tissue deposition and degradation.

Neurons forming optic glomeruli compute figure-ground discriminations in Drosophila.

Many animals rely on visual figure-ground discrimination to aid in navigation, and to draw attention to salient features like conspecifics or predators. Even figures that are similar in pattern and luminance to the visual surroundings can be distinguished by the optical disparity generated by their relative motion against the ground, and yet the neural mechanisms underlying these visual discriminations are not well understood. We show in flies that a diverse array of figure-ground stimuli containing a motion-defined edge elicit statistically similar behavioral responses to one another, and statistically distinct behavioral responses from ground motion alone. From studies in larger flies and other insect species, we hypothesized that the circuitry of the lobula--one of the four, primary neuropiles of the fly optic lobe--performs this visual discrimination. Using calcium imaging of input dendrites, we then show that information encoded in cells projecting from the lobula to discrete optic glomeruli in the central brain group these sets of figure-ground stimuli in a homologous manner to the behavior; "figure-like" stimuli are coded similar to one another and "ground-like" stimuli are encoded differently. One cell class responds to the leading edge of a figure and is suppressed by ground motion. Two other classes cluster any figure-like stimuli, including a figure moving opposite the ground, distinctly from ground alone. This evidence demonstrates that lobula outputs provide a diverse basis set encoding visual features necessary for figure detection.

Role of miR-155 in the pathogenesis of herpetic stromal keratitis.

Ocular infection with herpes simplex virus 1 can result in a chronic immunoinflammatory stromal keratitis (SK) lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage and to manipulate them therapeutically. Potential targets for therapy are miRNAs, but these are minimally explored especially in responses to infection. Here, we demonstrated that Mir155 expression was up-regulated after ocular herpes simplex virus 1 infection, with the increased Mir155 expression occurring mainly in macrophages and CD4(+) T cells and to a lesser extent in neutrophils. In vivo studies indicated that Mir155 knockout mice were more resistant to herpes SK with marked suppression of T helper cells type 1 and 17 responses both in the ocular lesions and the lymphoid organs. The reduced SK lesion severity was reflected by increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 and interferon-γ receptor α-chain levels in activated CD4(+) T cells in the lymph nodes. Finally, in vivo silencing of miR-155 by the provision of antagomir-155 nanoparticles to herpes simplex virus 1-infected mice led to diminished SK lesions and corneal vascularization. In conclusion, our results indicate that miR-155 contributes to the pathogenesis of SK and represents a promising target to control SK severity.

Olfactory neuromodulation of motion vision circuitry in Drosophila.

It is well established that perception is largely multisensory; often served by modalities such as touch, vision, and hearing that detect stimuli emanating from a common point in space; and processed by brain tissue maps that are spatially aligned. However, the neural interactions among modalities that share no spatial stimulus domain yet are essential for robust perception within noisy environments remain uncharacterized. Drosophila melanogaster makes its living navigating food odor plumes. Odor acts to increase the strength of gaze-stabilizing optomotor reflexes to keep the animal aligned within an invisible plume, facilitating odor localization in free flight. Here, we investigate the cellular mechanism for cross-modal behavioral interactions. We characterize a wide-field motion-selective interneuron of the lobula plate that shares anatomical and physiological similarities with the "Hx" neuron identified in larger flies. Drosophila Hx exhibits cross-modal enhancement of visual responses by paired odor, and presynaptic inputs to the lobula plate are required for behavioral odor tracking but are not themselves the target of odor modulation, nor is the neighboring wide-field "HSE" neuron. Octopaminergic neurons mediating increased visual responses upon flight initiation also show odor-evoked calcium modulations and form connections with Hx dendrites. Finally, restoring synaptic vesicle trafficking within the octopaminergic neurons of animals carrying a null mutation for all aminergic signaling is sufficient to restore odor-tracking behavior. These results are the first to demonstrate cellular mechanisms underlying visual-olfactory integration required for odor localization in fruit flies, which may be representative of adaptive multisensory interactions across taxa.

Therapy of respiratory viral infections with intranasal siRNAs.

Chemically synthesized short interfering RNA (siRNA) has ushered a new era in the application of RNA interference (RNAi) against viral genes. We have paid particular attention to respiratory viruses that wreak heavy morbidity and mortality worldwide. The clinically significant ones include respiratory syncytial virus (RSV), parainfluenza virus (PIV) (two Paramyxoviruses), and influenza virus (an Orthomyxovirus). As the infection by these viruses is clinically restricted to the respiratory tissues, mainly the lungs, the logical route for the application of the siRNA was also the same, i.e., via the nasal route. Following the initial success of single intranasal siRNA against RSV, we now offer two new strategies: (1) second-generation siRNAs, used against the paramyxoviral RNA polymerase large subunit (L), (2) siRNA cocktail with a novel transfection reagent, used against influenza virus. Based on these results, we propose the following consensus for designing intranasal antiviral siRNAs: (a) modified 19-27 nt-long double-stranded siRNAs are functional in the lung, (b) excessive 2'-OMe and 2'-F modifications in either or both strands of these siRNAs reduce efficacy, (c) limited modifications in the sense strand are beneficial, although their precise efficacy may be position-dependent, (d) cocktail of multiple siRNAs can be highly effective against multiple viral strains and subtypes.

Method and software for using m-sequences to characterize parallel components of higher-order visual tracking behavior in Drosophila.

A moving visual figure may contain first-order signals defined by variation in mean luminance, as well as second-order signals defined by constant mean luminance and variation in luminance envelope, or higher-order signals that cannot be estimated by taking higher moments of the luminance distribution. Separating these properties of a moving figure to experimentally probe the visual subsystems that encode them is technically challenging and has resulted in debated mechanisms of visual object detection by flies. Our prior work took a white noise systems identification approach using a commercially available electronic display system to characterize the spatial variation in the temporal dynamics of two distinct subsystems for first- and higher-order components of visual figure tracking. The method relied on the use of single pixel displacements of two visual stimuli according to two binary maximum length shift register sequences (m-sequences) and cross-correlation of each m-sequence with time-varying flight steering measurements. The resultant spatio-temporal action fields represent temporal impulse responses parameterized by the azimuthal location of the visual figure, one STAF for first-order and another for higher-order components of compound stimuli. Here we review m-sequence and reverse correlation procedures, then describe our application in detail, provide Matlab code, validate the STAFs, and demonstrate the utility and robustness of STAFs by predicting the results of other published experimental procedures. This method has demonstrated how two relatively modest innovations on classical white noise analysis--the inclusion of space as a way to organize response kernels and the use of linear decoupling to measure the response to two channels of visual information simultaneously--could substantially improve our basic understanding of visual processing in the fly.

Oligonucleotide aptamers: new tools for targeted cancer therapy.

Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed "chemical antibodies." In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

Flies dynamically anti-track, rather than ballistically escape, aversive odor during flight.

Tracking distant odor sources is crucial to foraging, courtship and reproductive success for many animals including fish, flies and birds. Upon encountering a chemical plume in flight, Drosophila melanogaster integrates the spatial intensity gradient and temporal fluctuations over the two antennae, while simultaneously reducing the amplitude and frequency of rapid steering maneuvers, stabilizing the flight vector. There are infinite escape vectors away from a noxious source, in contrast to a single best tracking vector towards an attractive source. Attractive and aversive odors are segregated into parallel neuronal pathways in flies; therefore, the behavioral algorithms for avoidance may be categorically different from tracking. Do flies plot random ballistic or otherwise variable escape vectors? Or do they instead make use of temporally dynamic mechanisms for continuously and directly avoiding noxious odors in a manner similar to tracking appetitive ones? We examine this question using a magnetic tether flight simulator that permits free yaw movements, such that flies can actively orient within spatially defined odor plumes. We show that in-flight aversive flight behavior shares all of the key features of attraction such that flies continuously 'anti-track' the noxious source.

Role of miR-132 in angiogenesis after ocular infection with herpes simplex virus.

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.