In vivo hamster cheek pouch subepithelial ablation, biomaterial injection, and localization: pilot study.

SIGNIFICANCE: The creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring. AIM: We aim to prove that subepithelial voids can be created in a live animal model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids. APPROACH: A benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention. RESULTS: Focused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation. CONCLUSIONS: We successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live animal model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large animals will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.

Outracing champion Gran Turismo drivers with deep reinforcement learning.

Many potential applications of artificial intelligence involve making real-time decisions in physical systems while interacting with humans. Automobile racing represents an extreme example of these conditions; drivers must execute complex tactical manoeuvres to pass or block opponents while operating their vehicles at their traction limits1. Racing simulations, such as the PlayStation game Gran Turismo, faithfully reproduce the non-linear control challenges of real race cars while also encapsulating the complex multi-agent interactions. Here we describe how we trained agents for Gran Turismo that can compete with the world's best e-sports drivers. We combine state-of-the-art, model-free, deep reinforcement learning algorithms with mixed-scenario training to learn an integrated control policy that combines exceptional speed with impressive tactics. In addition, we construct a reward function that enables the agent to be competitive while adhering to racing's important, but under-specified, sportsmanship rules. We demonstrate the capabilities of our agent, Gran Turismo Sophy, by winning a head-to-head competition against four of the world's best Gran Turismo drivers. By describing how we trained championship-level racers, we demonstrate the possibilities and challenges of using these techniques to control complex dynamical systems in domains where agents must respect imprecisely defined human norms.

Ultrafast laser surgery probe with a calcium fluoride miniaturized objective for bone ablation.

We present a miniaturized ultrafast laser surgery probe with improved miniaturized optics to deliver higher peak powers and enable higher surgical speeds than previously possible. A custom-built miniaturized CaF2 objective showed no evidence of the strong multiphoton absorption observed in our previous ZnS-based probe, enabling higher laser power delivery to the tissue surface for ablation. A Kagome fiber delivered ultrashort pulses from a high repetition rate fiber laser to the objective, producing a focal beam radius of 1.96 µm and covering a 90×90 µm2 scan area. The probe delivered the maximum available fiber laser power, providing fluences >6 J/cm2 at the tissue surface at 53% transmission efficiency. We characterized the probe's performance through a parametric ablation study on bovine cortical bone and defined optimal operating parameters for surgery using an experimental- and simulation-based approach. The entire opto-mechanical system, enclosed within a 5-mm diameter housing with a 2.6-mm diameter probe tip, achieved material removal rates >0.1 mm3/min, however removal rates were ultimately limited by the available laser power. Towards a next generation surgery probe, we simulated maximum material removal rates when using a higher power fiber laser and found that removal rates >2 mm3/min could be attained through appropriate selection of laser surgery parameters. With future development, the device presented here can serve as a precise surgical tool with clinically viable speeds for delicate applications such as spinal decompression surgeries.

Pharmaceutical industry-authored preprints: scientific and social media impact.

AIM: Non-peer-reviewed manuscripts posted as preprints can be cited in peer-reviewed articles, which has both merits and demerits. International Committee of Medical Journal Editors guidelines mandate authors to declare preprints at the time of manuscript submission. We evaluated the trends in pharma-authored research published as preprints and their scientific and social media impact by analyzing citation rates and altmetrics. RESEARCH DESIGN AND METHODS: We searched EuroPMC, PrePubMed bioRxiv and medRxiv for preprints submitted by authors affiliated with the top 50 pharmaceutical companies from inception until 15 June 2020. Data were extracted and analyzed from the search results. The number of citations for the preprint and peer-reviewed versions (if available) were compiled using the Publish or Perish software (version 1.7). Altmetric score was calculated using the "Altmetric it" online tool. Statistical significance was analyzed by Wilcoxon rank-sum test. RESULTS: A total of 498 preprints were identified across bioRxiv (83%), PeerJ (5%), F1000Research (6%), Nature Precedings (3%), Preprint.org (3%), Wellcome Open Research preprint (0.2%) and medRxiv (0.2%) servers. Roche, Sanofi and Novartis contributed 56% of the retrieved preprints. The median number of citations for the included preprints was 0 (IQR = 1, Min-Max = 0-45). The median number of citations for the published preprints and unpublished preprints was 0 for both (IQR = 1, Min-Max = 0-25 and IQR = 1, Min-Max = 0-45, respectively; p = .091). The median Altmetric score of the preprints was 4 (IQR = 10.5, Min-Max = 0-160). CONCLUSION: Pharma-authored research is being increasingly published as preprints and is also being cited in other peer-reviewed publications and discussed in social media.

Perspectives on the Early Quality of Evidence Guiding the Therapeutic Management of SARS-CoV-2: A Systematic Literature Review.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak is a serious health concern. Repurposing of existing drugs indicated for other conditions seems to be the first choice for immediate therapeutic management. The quality of early evidence favoring the different treatment options needs to be apprised for informed decision-making. METHODS: In this systematic literature review, we apprised the quality of available evidence for different therapeutic options and also the basis for different treatment guidelines. To include all studies that are in different stages of publication, we also included studies from the preprint servers BioRxiv and MedRxiv and published studies from PubMed. RESULTS: We retrieved 5621 articles and included 22 studies for the systematic review. Based on our study, chloroquine/hydroxychloroquine, either alone or in combination with azithromycin, remdesivir, corticosteroids, convalescent sera, ritonavir/lopinavir, tocilizumab and arbidol were evaluated as therapeutic options. The data from different study designs reveal contradictory findings except for convalescent sera for which the evidence available is only from case series. Based on this early evidence, various national guidelines recommend remdesivir, convalescent sera, corticosteroids and hydroxychloroquine in different subsets of patients. CONCLUSION: Establishing consensus with respect to the end points to be assessed for respiratory viruses may enhance the quality of evidence in case of future pandemics. The systematic review highlighted the lacuna and methodologic deficiency in early clinical evidence and included an update on different therapeutic management guidelines. Further clinical evidence from the ongoing trials may lead to evolution of treatment guidelines with the addition of more therapeutic options.

Femtosecond Plasmonic Laser Nanosurgery (fs-PLN) mediated by molecularly targeted gold nanospheres at ultra-low pulse fluences.

Plasmonic Laser Nanosurgery (PLN) is a novel photomodification technique that exploits the near-field enhancement of femtosecond (fs) laser pulses in the vicinity of gold nanoparticles. While prior studies have shown the advantages of fs-PLN to modify cells, further reduction in the pulse fluence needed to initiate photomodification is crucial to facilitate deep-tissue treatments. This work presents an in-depth study of fs-PLN at ultra-low pulse fluences using 47 nm gold nanoparticles, conjugated to antibodies that target the epithelial growth factor receptor and excited off-resonance using 760 nm, 270 fs laser pulses at 80 MHz repetition rate. We find that fs-PLN can optoporate cellular membranes with pulse fluences as low as 1.3 mJ/cm2, up to two orders of magnitude lower than those used at lower repetition rates. Our results, corroborated by simulations of free-electron generation by particle photoemission and photoionization of the surrounding water, shed light on the off-resonance fs-PLN mechanism. We suggest that photo-chemical pathways likely drive cellular optoporation and cell damage at these off-resonance, low fluence, and high repetition rate fs-laser pulses, with clusters acting as local concentrators of ROS generation. We believe that the low fluence and highly localized ROS-mediated fs-PLN approach will enable targeted therapeutics and cancer treatment.

Kagome fiber based ultrafast laser microsurgery probe delivering micro-Joule pulse energies.

We present the development of a 5 mm, piezo-actuated, ultrafast laser scalpel for fast tissue microsurgery. Delivery of micro-Joules level energies to the tissue was made possible by a large, 31 µm, air-cored inhibited-coupling Kagome fiber. We overcome the fiber's low NA by using lenses made of high refractive index ZnS, which produced an optimal focusing condition with 0.23 NA objective. The optical design achieved a focused laser spot size of 4.5 µm diameter covering a 75 × 75 µm2 scan area in a miniaturized setting. The probe could deliver the maximum available laser power, achieving an average fluence of 7.8 J/cm2 on the tissue surface at 62% transmission efficiency. Such fluences could produce uninterrupted, 40 µm deep cuts at translational speeds of up to 5 mm/s along the tissue. We predicted that the best combination of speed and coverage exists at 8 mm/s for our conditions. The onset of nonlinear absorption in ZnS, however, limited the probe's energy delivery capabilities to 1.4 µJ for linear operation at 1.5 picosecond pulse-widths of our fiber laser. Alternatives like broadband CaF2 crystals should mitigate such nonlinear limiting behavior. Improved opto-mechanical design and appropriate material selection should allow substantially higher fluence delivery and propel such Kagome fiber-based scalpels towards clinical translation.

Intra Luminal Metastasis to Duodenum: A Histological Surprise.

Carcinoma cervix is one of the most common malignancies seen in women worldwide and more so in the Indian subcontinent. Carcinoma cervix is known for its orderly lymphatic spread. Skip metastasis or intraluminal metastasis is not a frequent occurrence. A 50-year-old diabetic and hypertensive female patient who was diagnosed to have carcinoma cervix (stage II A 2) Grade II to III and post radiotherapy (3 years back) presented to the surgical outpatient with dyspeptic symptoms and vague abdominal pain. On examination she was found to have a 1x1cm hard, fixed, left supraclavicular node and a palpable liver. Ultrasound abdomen revealed multiple retro peritoneal lymphadenopathy and Contrast Enhanced Computerised Tomography (CECT) abdomen revealed secondary deposits in the pancreatico-duodenal groove infiltrating the duodenal C-loop and pancreatic head, with multiple retroperitoneal nodes with necrosis. Fine needle Aspiration Cytology (FNAC) from left supraclavicular node revealed metastatic squamous cell carcinoma. Oesophagogastroduodenoscopy showed 2 sessile lesions in the anterior wall of duodenum with mucosa stretched and central umbilication with almost complete luminal obstruction. Biopsy from the duodenal lesion also turned out positive for metastatic squamous cell carcinoma. Gynaecological examination was negative for any local recurrence. Patient was managed symptomatically for dyspepsia and is currently undergoing chemotherapy. She is at present on regular follow-up and is asymptomatic for the bowel metastasis. Given the frequency of cancer cervix and the rarity of intra luminal metastasis, this case report serves to reiterate the fact that the abdomen is truly a pandora's box.

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The blaNDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: blaNDM-1 in 2 isolates, blaNDM-5 in 7 isolates, and blaNDM-7 in 5 isolates. The blaCTX-M gene was detected in 112 isolates (66.3%): blaCTX-M-15 in 108 isolates and blaCTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the blaNDM-positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

Design and fabrication of a miniature objective consisting of high refractive index zinc sulfide lenses for laser surgery.

A miniature laser ablation probe relying on an optical fiber to deliver light requires a high coupling efficiency objective with sufficient magnification in order to provide adequate power and field for surgery. A diffraction-limited optical design is presented that utilizes high refractive index zinc sulfide to meet specifications while reducing the miniature objective down to two lenses. The design has a hypercentric conjugate plane on the fiber side and is telecentric on the tissue end. Two versions of the objective were built on a diamond lathe-a traditional cylindrical design and a custom-tapered mount. Both received an antireflective coating. The objectives performed as designed in terms of observable resolution and field of view as measured by imaging a 1951 USAF resolution target. The slanted edge technique was used to find Strehl ratios of 0.75 and 0.78, respectively, indicating nearly diffraction-limited performance. Finally, preliminary ablation experiments indicated threshold fluence of gold film was comparable to similar reported probes.

A 5-mm piezo-scanning fiber device for high speed ultrafast laser microsurgery.

Towards developing precise microsurgery tools for the clinic, we previously developed image-guided miniaturized devices using low repetition rate amplified ultrafast lasers for surgery. To improve the speed of tissue removal while reducing device diameter, here we present a new 5-mm diameter device that delivers high-repetition rate laser pulses for high speed ultrafast laser microsurgery. The device consists of an air-core photonic bandgap fiber (PBF) for the delivery of high energy pulses, a piezoelectric tube actuator for fiber scanning, and two aspheric lenses for focusing the light. Its inline optical architecture provides easy alignment and substantial size reduction to 5 mm diameter as compared to our previous MEMS-scanning devices while realizing improved intensity squared (two-photon) lateral and axial resolutions of 1.16 µm and 11.46 µm, respectively. Our study also sheds light on the maximum pulse energies that can be delivered through the air-core PBF and identifies cladding damage at the input facet of the fiber as the limiting factor. We have achieved a maximum energy delivery larger than 700 nJ at 92% coupling efficiency. An in depth analysis reveals how this value is greatly affected by possible slight misalignments of the beam during coupling and the measured small beam pointing fluctuations. In the absence of these imperfections, self-phase modulation becomes the limiting factor for the maximum energy delivery, setting the theoretical upper bound to near 2 µJ for a 1-m long, 7-µm, air-core PBF. Finally, the use of a 300 kHz repetition rate fiber laser enabled rapid ablation of 150 µm x 150 µm area within only 50 ms. Such ablation speeds can now allow the surgeons to translate the surgery device as fast as ~4 mm/s to continuously remove a thin layer of a 150 µm wide tissue. Thanks to a high optical transmission efficiency of the in-line optical architecture of the device and improved resolution, we could successfully perform ablation of scarred cheek pouch tissue, drilling through a thin slice. With further development, this device can serve as a precise and high speed ultrafast laser scalpel in the clinic.