Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model ...for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle
. The model comprises four (meta)stable intermediates (S
, S
, S
and S
) and one transient S
state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn
CaO
) cluster in the oxygen-evolving complex
. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q
at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S
→S
state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S
state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O
release. Thus, our results exclude peroxo-bond formation in the S
state, and the nucleophilic attack of W3 onto W2 is unlikely.
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional ...control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.
Remote digital pathology allows healthcare systems to maintain pathology operations during public health emergencies. Existing Clinical Laboratory Improvement Amendments regulations require ...pathologists to electronically verify patient reports from a certified facility. During the 2019 pandemic of COVID-19 disease, caused by the SAR-CoV-2 virus, this requirement potentially exposes pathologists, their colleagues, and household members to the risk of becoming infected. Relaxation of government enforcement of this regulation allows pathologists to review and report pathology specimens from a remote, non-CLIA certified facility. The availability of digital pathology systems can facilitate remote microscopic diagnosis, although formal comprehensive (case-based) validation of remote digital diagnosis has not been reported. All glass slides representing routine clinical signout workload in surgical pathology subspecialties at Memorial Sloan Kettering Cancer Center were scanned on an Aperio GT450 at ×40 equivalent resolution (0.26 µm/pixel). Twelve pathologists from nine surgical pathology subspecialties remotely reviewed and reported complete pathology cases using a digital pathology system from a non-CLIA certified facility through a secure connection. Whole slide images were integrated to and launched within the laboratory information system to a custom vendor-agnostic, whole slide image viewer. Remote signouts utilized consumer-grade computers and monitors (monitor size, 13.3–42 in.; resolution, 1280 × 800–3840 × 2160 pixels) connecting to an institution clinical workstation via secure virtual private network. Pathologists subsequently reviewed all corresponding glass slides using a light microscope within the CLIA-certified department. Intraobserver concordance metrics included reporting elements of top-line diagnosis, margin status, lymphovascular and/or perineural invasion, pathology stage, and ancillary testing. The median whole slide image file size was 1.3 GB; scan time/slide averaged 90 s; and scanned tissue area averaged 612 mm2. Signout sessions included a total of 108 cases, comprised of 254 individual parts and 1196 slides. Major diagnostic equivalency was 100% between digital and glass slide diagnoses; and overall concordance was 98.8% (251/254). This study reports validation of primary diagnostic review and reporting of complete pathology cases from a remote site during a public health emergency. Our experience shows high (100%) intraobserver digital to glass slide major diagnostic concordance when reporting from a remote site. This randomized, prospective study successfully validated remote use of a digital pathology system including operational feasibility supporting remote review and reporting of pathology specimens, and evaluation of remote access performance and usability for remote signout.
While COVID-19 significantly overburdens emergency rooms (ERs) and hospitals in affected areas, ERs elsewhere report a marked decrease in patient numbers. This study aimed to investigate the ...assumption that patients with urgent problems currently avoid the ER.
Electronic health records from the ER of a large Swiss university hospital were extracted for three periods: first, the awareness phase (ap) from the publication of the national government's initiative "How to protect ourselves" on 1 March 2020 to the lockdown of the country on 16 March; second, the mitigation phase (mp) from 16-30 March; finally, patients presenting in March 2019 were used as a control group. We compared parameters including a critical illness as the discharge diagnosis (e.g., myocardial infarction, stroke, sepsis and ER death) using logistic and linear regression, as well as 15-day bootstrapped means and 95% confidence intervals for the control group.
In the three periods, a total of 7143 patients were treated. We found a 24.9% (42.5%) significant decline in the number of patients presenting during the ap (mp). Patients presenting during the mp were more likely to be critically ill. There was an increase of 233% and 367% (ap and mp, respectively) of ER deaths (none related to COVID-19) compared with the control period. Apart from polytrauma (increase of 5% in the mp), all other critical illnesses as discharge diagnosis showed a lower incidence in descriptive analysis. Significantly more patients died in the ER in both the ap and mp.
Barriers to seeking emergency care during COVID-19 pandemic may lead to higher morbidity and mortality. Healthcare authorities and hospitals must ensure low barriers to treatment and business as usual for all patients.
Poor limb conformation in cattle is believed to be closely associated with claw and limb disorders. Limb conformation is part of genetic evaluation and is assessed visually. In this descriptive ...study, the assessment of conformation in calves was evaluated objectively using joint angle measurements. A total of 100, 30- to 90-d-old, heifer calves of the Brown Swiss (n = 50) and Holstein (n = 50) breeds were photographed from both sides and the front and rear. Carpal, tarsal, autopodium and fetlock joint angles of the fore- or hind limbs were measured on the images using anatomic reference points and the ImageJ/Fiji® program. Each angle was measured 3 times, and the mean was used for analysis. Values from both sides were pooled. Deviations from defined standard angles were classified as slight or moderate. The positions of the front and hind feet were measured and scored. On average, the calves had moderate „knock-kneed“ carpal conformation, and the autopodium of the front limb had a slight lateral deviation. In the rear view, the calves had slight, but close to moderate, „cow-hocked“ tarsal conformation, and the autopodium of the hind limbs was parallel, but close to slightly deviated. Holstein calves were moderately, and Brown Swiss calves were slightly „cow-hocked“. A moderate lateral deviation occurred in the metacarpal-digit angle in the front view and a slight lateral deviation occurred in the metatarsal-digit angle in the rear view. The front feet position score was >17° in 69% of the calves, and the hind feet position score was <17° in 90% of the calves. In the side views, the calves were slightly “over at the carpus” and slightly “straight-hocked” and had average fetlock conformation.
In the mean values, most measured joint angles did not correspond to the straight or parallel classifications in our calves; moderate „knock-kneed“ and slight „cow-hocked“, and “toed-out” classifications were most common. The limb angles measured were based on anatomical features and therefore are unlikely to change substantially with increasing age. These aspects should be considered when assessing linear traits and investigating the relationship between limb conformation and claw diseases.
Inorganic-organic hybrid materials represent a large share of newly reported structures, owing to their simple synthetic routes and customizable properties
. This proliferation has led to a ...characterization bottleneck: many hybrid materials are obligate microcrystals with low symmetry and severe radiation sensitivity, interfering with the standard techniques of single-crystal X-ray diffraction
and electron microdiffraction
. Here we demonstrate small-molecule serial femtosecond X-ray crystallography (smSFX) for the determination of material crystal structures from microcrystals. We subjected microcrystalline suspensions to X-ray free-electron laser radiation
and obtained thousands of randomly oriented diffraction patterns. We determined unit cells by aggregating spot-finding results into high-resolution powder diffractograms. After indexing the sparse serial patterns by a graph theory approach
, the resulting datasets can be solved and refined using standard tools for single-crystal diffraction data
. We describe the ab initio structure solutions of mithrene (AgSePh)
, thiorene (AgSPh) and tethrene (AgTePh), of which the latter two were previously unknown structures. In thiorene, we identify a geometric change in the silver-silver bonding network that is linked to its divergent optoelectronic properties
. We demonstrate that smSFX can be applied as a general technique for structure determination of beam-sensitive microcrystalline materials at near-ambient temperature and pressure.
Chimeric antigen receptor (CAR) T cells have emerged as a potent therapeutic approach for patients with certain haematological cancers, with multiple CAR T cell products currently approved by the FDA ...for those with relapsed and/or refractory B cell malignancies. However, in order to derive the desired level of effectiveness, patients need to successfully receive the CAR T cell infusion in a timely fashion. This process entails apheresis of the patient's T cells, followed by CAR T cell manufacture. While awaiting infusion at an authorized treatment centre, patients may receive interim disease-directed therapy. Most patients will also receive a course of pre-CAR T cell lymphodepletion, which has emerged as an important factor in enabling durable responses. The time between apheresis and CAR T cell infusion is often not a simple journey, with each milestone being a critical step that can have important downstream consequences for the ability to receive the infusion and the strength of clinical responses. In this Review, we provide a summary of the many considerations for preparing patients with B cell non-Hodgkin lymphoma or acute lymphoblastic leukaemia for CAR T cell therapy, and outline current limitations and areas for future research.
Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X‐ray absorption edges, which shift to higher energy for progressively more positive valence numbers. ...Indeed, X‐ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X‐ray sources at synchrotron facilities with energy resolutions in the range 2–3 × 10−4 (ΔE/E). This paper tests whether X‐ray free‐electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10−3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength‐dependent anomalous scattering factors contributed by two differently charged iron centers in the 110‐amino‐acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X‐ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.
Electronic configurations at distinct metal centers within a metalloprotein may be characterized by inspecting the scattering factors at the X‐ray absorption edge. Such experiments may be feasible at XFEL sources, using Bayesian data analysis.
Immune checkpoint inhibitors (ICIs) are standard therapies for patients with advanced non-small-cell lung cancer (NSCLC) and a programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) ≥50%. ...Tumor mutation burden (TMB) also predicts response to ICIs but is often not available in real time for decision making in the first-line setting. Smoking exposure can be a proxy for TMB in NSCLC. The impact of smoking status on efficacy of PD-1 blockade in NSCLC patients with PD-L1 TPS ≥50% has not been well defined.
To investigate the relationship between smoking and activity of ICIs in NSCLC, we retrospectively studied 315 patients with NSCLC and PD-L1 TPS ≥50% at five USA academic medical centers. Objective response rates (ORRs), progression-free survival (PFS), and duration of response (DOR) were compared between never (<100 lifetime cigarettes), light (≤10 pack-years), and heavy (>10 pack-years) smokers. A subset of patients underwent next-generation sequencing to estimate TMB.
We identified 36 (11%) never, 42 (13%) light, and 237 (75%) heavy smokers with NSCLC and PD-L1 TPS ≥50% treated with ICIs. Objective responses were observed in 27%, 40%, and 40% of never, light, and heavy smokers, respectively (P = 0.180 never versus heavy; P = 1.000 light versus heavy). Median PFS and median DOR were numerically shorter in never and light smokers compared with heavy smokers (PFS 3.0 versus 4.0 versus 5.4 months; median DOR 6.9 versus 10.8 versus 17.8 months), but were not statistically different PFS: hazard ratio (HR) 1.37, P = 0.135 and HR 1.24, P = 0.272; DOR: HR 1.92, P = 0.217 and HR 1.79, P = 0.141.
PD-(L)1 inhibitors are associated with antitumor activity in NSCLC with PD-L1 TPS ≥50% regardless of smoking status. Nevertheless, there is a signal of potentially decreased durability among never and light smokers that should be further evaluated. Distinct immunobiologic features may affect initial response versus durability of antitumor immunity to programmed cell death 1 (PD-1) blockade.
•In patients with NSCLC and PD-L1 ≥50%, TMB is lower in never and light smokers compared with heavy smokers.•PD-(L)1 inhibitors are active in a significant subset of never and light smokers with NSCLC and PD-L1 ≥50%.•Nonetheless, we observed numerically shorter PFS/DOR among never and light smokers compared to heavy smokers.•Distinct immunobiologic features may underlie initial response versus durability of response to PD-1 inhibitors in NSCLC.
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