Based on ACL-RSI scores, they were divided into “cases” and “controls”, deemed not psychologically ready and psychologically ready to return to previous sport performance (PILOS), respectively. The main findings are that quadriceps’ and hamstrings’ rate of torque development (RTD) and time since surgery were determinants of psychological readiness following ACLR. Furthermore, compared to controls, cases showed significantly lower quadriceps torque at angles close to full knee extension (40 deg and 30 deg from extension). They also showed lower RTD than controls, but no difference in peak torque. These results suggest that physiotherapists should facilitate athletes’ return to sport (RTS) by focusing on the restoration of RTD and strength at angles close to full knee extension.Psychological readiness following anterior cruciate ligament reconstruction (ACLR) correlates with different return to sport outcomes. However, the relationship between strength and power and psychological readiness remains unexplored. The aim of this study was to investigate the relationship between anterior cruciate ligament return to sport after injury (ACL-RSI) scores and various hamstrings and quadriceps strength and power variables. Twelve participants  who had an ACLR nine months or more before the study completed the ACL-RSI questionnaire and isokinetic strength testing of the hamstrings and quadriceps (60°·s Anterior cruciate ligament injury (ACLI) is a common and incapacitating knee injury often sustained during sport participation and treated with ACL reconstruction (ACLR) followed by rehabilitation . Return While the reasons why an athlete may not resume participation to their PILOS are multifactorial, the role played by psychological factors seems crucial . Indeed,ecc) and quadriceps concentric peak torque (Qcon). Other characteristics of these athletes that might put them more at risk of ACLI include a low limb symmetry index (LSI)  × 100.The following variables were calculated as the average of the two best contractions at 60°·s−1 vs. 180°·s−1) on all outcome variables. Effect sizes were calculated as partial eta squared (ƞ2p) for the ANOVA and interpreted as no effect (0–0.05), minimum effect (0.05–0.26), and strong effect (0.26–0.64), while Cohen’s d (d) represented the effect size for post-hoc tests, and were interpreted as small (>0.2), medium (>0.5), and large (>0.8), [p-value was set at p < 0.05. All data were presented as mean ± standard deviation, with a 95% confidence interval (CI). Statistical analyses were conducted using IBM SPSS V.29. The normality of data was assessed using the Shapiro–Wilk test. A stepwise linear regression was conducted on the entire sample to identify if strength data from the operated leg could be determinants of the ACL-RSI score. Subsequently, participants were divided into a ready to return to PILOS group and not ready to return to PILOS group. A mixed model analysis of variance (ANOVA) with repeated measures was performed to assess differences between groups (ready vs. not ready to return to PILOS) and angular velocities (60°·s (>0.8), . The p-vn = 4), volleyball (n = 2), netball (n = 2), lacrosse (n = 2), tennis (n = 1), and triathlon (n = 1). Seven participants were identified as cases with ACL-RSI scores <81.4% and five participants were identified as controls with ACL-RSI scores > 81.5. Participant Demographics are summarised in We recruited participants from football . The statistics associated with each factor were beta = 0.583, t = 5.077 (p < 0.001) for quadriceps RTD100 at 60°·s−1; beta = 0.620, t = 5.855 (p < 0.001) for time since surgery; and beta = 0.411, t = 3.498 (p = 0.008) for hamstrings RTD50 at 60°·s−1.The linear regression identified that three variables, namely quadriceps RTDcon and Hecc. Unfortunately, we did not get enough data in the 0–10° range due to a lack of flexibility of some of our participants, and hence, these were removed from the analysis. The ANOVA showed a significant effect of angular velocity on Qcon peak torque (F(df) = 98.529(1), p = 0.001, ƞ2p = 0.908), Qcon40 (F(df) = 23.167(1), p = 0.001, ƞ2p = 0.698), Hecc30 (F(df) = 7.012(1), p = 0.024, ƞ2p = 0.412), and Hecc20 (F(df) = 5.817(1), p = 0.039, ƞ2p = 0.393). Post-hoc analyses revealed significantly greater values at 60°·s−1 compared to 180°·s−1 in Qcon peak torque  = 4.975(1), p = 0.048, ƞ2p = 0.345) and Qcon30 (F(df) = 4.445(1), p = 0.049, ƞ2p = 0.410). Significantly lower values were observed in cases compared to controls in Qcon40 .ecc RTD50 (F(df) = 20.107(1), p = 0.001, ƞ2p = 0.668) and Hecc RTD100 (F(df) = 20.169(1), p = 0.001, ƞ2p = 0.669). Post-hoc analyses revealed significantly lower values at 60°·s−1 compared to 180°·s−1 on Hecc RTD50  = 4.679(1), p = 0.048, ƞ2p = 0.338), with significantly lower values observed in cases compared to controls .The statistical analysis showed a significant effect of angular velocity on Hcon RTD100 (F(df) = 4.589(1), p = 0.045, ƞ2p = 0.243) and LSI Hecc RTD50 (F(df) = 6.224(1), p = 0.032, ƞ2p = 0.384). Significantly greater values were observed in cases compared to controls on LSI Qcon RTD100  and significantly lower values were observed in cases compared to controls on LSI Hecc RTD100 . We did not find any significant interaction between groups and velocities on any LSI variables (p > 0.05).Regarding asymmetry between limbs, the ANOVA showed a significant group effect on LSI Qcon close to extension and lower RTD for Qcon, but no difference in peak torque values. This study is one of very few observing the relationship between strength and power parameters and psychological readiness following ACLR. We measured a range of parameters during concentric contractions of the quadriceps and eccentric contractions of the hamstrings, since these antagonist muscles are involved in many sport movements. In particular, the hamstrings act as an ACL agonist by eccentrically contracting to decelerate the anterior translation of the tibia during powerful quadriceps contractions. The main findings are that the RTD of the quadriceps and hamstrings, as well as time since surgery, were the main determinants of psychological readiness following ACLR. In addition, compared to controls, cases showed significantly lower angle-specific Qecc:Qcon or high LSI [We recruited athletes mostly from team contact sports, despite advertising the study to all university sports. It is not very surprising due to the high occurrence of ACLI reported in these sports . It is whigh LSI ,11. A novel finding identified within this study is the extent to which RTD has been identified as both a determinant for psychological readiness and as a statistically significant difference between groups. This is the first study to examine the relationships between muscle RTD scores and psychological readiness, and, therefore, a comparison of results cannot be made against previous literature. The prevalence of RFD as a power deficit following ACLR, however, is well reported for the injured limb in the short term ,28 but acon at 60°·s−1 and ACL-RSI scores in athletes nine months post-surgery [ecc at 60°·s−1 and ACL-RSI scores [con or peak Hecc, in accordance with our results. The contrasting results on Hecc between the present study and the study of O’Connor et al. [n = 452) and their cut-off at nine months post-surgery, compared to the range of longitudinal time frames examined within this study. It can be hypothesised that athlete perceptions within O’Connor’s [The role of peak torque as a determinant of ACL-RSI scores was not identified in the present study, suggesting that maximal force production might not be as important as sport staff  or researchers thought. This is partly in line with the findings of the only study in this area reporting no significant relationship between peak Q-surgery . HoweverI scores , which iI scores  also sepr et al.  could ber et al.  comparedr et al. ’s study Connor’s  study arConnor’s , which mcon40 and Qcon30 in cases compared to controls . The role of the quadriceps at these shallow flexion angles is significant in relation to the number of ACLI that occur within them [ecc:Qcon was significantly reduced after a simulated match only between 0 and 10° from full knee extension [A novel finding of the present study is the significantly lower Qhin them . Similarhin them , yet lithin them ). Our fihin them , who simxtension . This hi100 in the quadriceps at both testing velocities was greater in cases than controls. This highlights one more time that power rather than strength could be an important factor that could play a role in athletes’ decisions to RTS. The limb symmetry index has been cited as a risk factor for lower limb injuries in previous literature , althougThe main limitations of the present study are the lack of control over our participants’ rehabilitation and subsequent sport experience. Indeed, we could not capture the exercise types, volume, and frequency of rehabilitation sessions or past/current sport participation due to the long time lapse between the start of rehabilitation and testing for some of our participants. In addition, we chose to focus on psychological parameters as our main outcome and explore the role of strength and power variables as potential determinants of this. However, an argument can be made that constructs such as fear and confidence may instead impact physiological parameters through the avoidance of high-intensity rehabilitation required in order to efficiently address them. Finally, our sample size was small, in particular in comparison with the study by O’Connor et al. , which cIn conclusion, our findings showed that one of the main determinants of psychological readiness to RTS following ACLR was the RTD of the quadriceps and hamstrings. In addition, compared to controls, cases showed significantly lower angle-specific quadriceps torque close to extension and lower quadriceps RTD, but no difference in peak torque values. The importance of muscular strength across all angles of function was also identified, with quadriceps strength of particular importance at angles of functional relevance between 20° and 40° from full extension. These findings highlight the importance for physiotherapists to address muscular deficits across their full range (by choosing some exercises with the knee joint positioned near full extension) and develop power and explosiveness in addition to strength in rehabilitation. Future studies with larger sample sizes are needed to further investigate the relationship between strength and power and psychological factors playing a role in RTS.


Salmorejo is a tomato–oil cold puree commercialized as a “fresh-like” product requiring mild pasteurisation and chill storage to reach a suitable shelf lifetime. The objective of this study was to study the factors which limit the shelf life of salmorejo pasteurised via conventional or radiofrequency continuous heating, packed in high-density polyethylene bottles, and kept at refrigeration. The pasteurised–chilled salmorejo reached a long shelf life (4 months) compared to that of pasteurised tomato juices or purees. Mesophilic and pathogenic bacteria were easily inhibited in this acidic product. Salmorejo mainly showed oxidative and subsequent sensory changes. Initial enzyme oxidation was associated with some adverse effects (loss of vitamin C and lipid oxidation) at the first month, although there were no sensory implications. Salmorejo remained stable at the physicochemical and sensory levels for the following 3 months, though colour and viscosity changes could be measured with instruments. Between the fourth and fifth month, salmorejo showed clear signs of deterioration, including changes in appearance (slight browning and loss of smooth surface), odour/flavour (loss of freshness and homogenisation), and consistency (thinning trend). The shelf life of salmorejo is limited by long-term oxidative deterioration and their sensory implications. Salmorejo is a Spanish traditional cold purée prepared with tomato, olive oil, breadcrumbs and other thickening ingredients, vinegar, garlic, and salt. The production of salmorejo and gazpacho (a similar product made with a different recipe in the same manufacturing lines) is growing in Spain (about 96 million litres in 2021), and it is now exported to other European countries . Packed Pasteurisation conditions reported for “fresh-like” gazpacho range from 80 to 90 °C for 1 min, resulting in products with a shelf life of 30–60 days in refrigeration . High-prL. monocytogenes. These HTST conditions also inactivated PME but did not completely inactivate PPO and POD and did not affect PG enzyme, while increasing the pasteurisation temperature up to 100 °C did not improve the results of enzyme inactivation, and there were some signs of overheating. This is of great relevance since “fresh-like” salmorejo should retain sensory traits like those of a fresh raw product for several months The research hypothesis was that HTST salmorejo mainly deteriorates due to oxidation phenomena enhanced, to a greater or lesser degree, by the tomato enzymes that remain active. The time that these enzymes can remain active in the pasteurised–chilled salmorejo has not yet been determined. Salmorejo presents some properties that might contribute to its oxidative stability: (i) tomato is rich in lycopene and other phenolic antioxidants; (ii) it is made with extra virgin olive oil rich in α-tocopherol and other lipid antioxidants; and (iii) this emulsion is thickened with breadcrumbs or other ingredients, which may delay clarification signs.Dielectric heating by radiofrequency (RF) has recently been tested to improve the pasteurisation methods for these types of tomato–oil emulsions. RF technology could replace conventional heat exchangers in the food industry as dielectric heating has better thermal penetration and could be less energy-intensive ,17. A prThe objective of this study was to study the factors which limit the shelf life of pasteurised–chilled salmorejo. Different microbiological, enzyme, physicochemical, and sensory aspects were studied in a salmorejo alternatively pasteurised via conventional (CH) or radiofrequency (RF) continuous heating, packed into HDPE bottles, and kept under refrigeration for up to five months.® high-polymerizate HP inulin, Beneo, Barcelona, Spain). Salmorejo ingredients (g·100 g−1) were vine tomatoes (87.6), extra virgin olive oil (Var. Hojiblanca) (5.0), breadcrumbs (3.0), inulin (2.0), wine vinegar (1.5), garlic (0.7), and salt (0.2). Briefly, tomatoes were ground in a cutter and sieved (3 mm diameter). Tomato purée was then mixed with olive oil, breadcrumbs, salt, vinegar, inulin, and garlic by stirring in a tank for 30 min. Raw salmorejo was homogenised in an MZ-100 colloid mill  and preheated at 50 °C for 15 s in a tubular heat exchanger. Before pasteurisation, preheated product passed through a high-pressure homogeniser , working at 250 bar. The thermal treatment was targeted to inactivate Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes to ensure at least a 5-log reduction in vegetative pathogens of concern. Applying 80 °C for around 10 s in a continuous line with a preheating stage provides an adequate decimal reduction in mesophilic bacteria and foodborne pathogens. This temperature was determined using the Z-values corresponding to an unacidified tomato purée at 4.5 pH. A minimum cumulative total lethality equivalent to p = 0.51 min at 71.1 °C was needed for a cocktail of pathogens 1/2. Dynamic viscosity was measured using two different procedures. Viscosity (V1) (Pa s) was measured in samples sheared for 1 min with a standard cylindrical vessel 125 × 28 mm  using a 6 L/plus rotational Viscotester . The measuring conditions for V1 were 80 mL sample; L3 probe; 100 rpm angular speed; 4 °C temperature. Viscosity (V2) (mPa s), including initial shear stress, was measured at 20 °C using an MV1 cylindrical probe in a Haake 550 rotational viscometer . Flow curves were obtained from stepped shear stress ramp (steady state approximation: 15 s per point). Ranges of shear stresses, in lineal distribution, were used to obtain shear rates between 450 and 1800 s−1. Flow curves data were fitted to the Bingham model (τ = τ0B + γμpl), where τ is the shear stress [Pa], γ˙ the shear rate [s−1], τ0B the Bingham yield stress [Pa], and μpl the plastic viscosity [Pa s]. Texture was analysed using a QTS-25 Analyser . Selected parameters were hardness (N), the maximum force required to compress the material, and deformation energy (mJ), the energy required to deform the sample. Operating conditions were 5 °C; TA3/100 flat cylindrical probe (20 mm in diameter); trigger point, 0.05 N; compression objective, 5 mm; crosshead speed, 0.1 mm s−1; and charge cell, 10 kg.The pH was measured with a GLP21 pHmeter  equipped with a combined electrode Cat. No. 52-21 . CIELab colour was measured using a CR-200/08 Chroma Meter II  with a D65 illuminant, 2° observer angle and 50 mm aperture size. Results were expressed as CIELab values: lightness (L*), redness (a*), yellowness (b*), hue angle  of the salmorejo samples was performed in 10 sessions/replica. The generation of the descriptors was carried out by open discussion and consensus in two previous sessions using both untreated and treated samples (CH and RF). Six selected and trained panellists  perfp < 0.05). Sample size was n = 108 (3 bottled samples of 250 mL × 2 heating methods × 6 retailing times × 3 manufacturing batches). Data were analysed using the Statistix 8.0 software for Windows .A randomised design was performed with the storage time (0–5 months) and heating method (CH or RF) as treatments. The effects of the treatments on the dependent variables were determined using a two-way ANOVA  and RF (3.90 Log·CFU g−1) untreated salmorejo. Log reductions in TMA counts reached through pasteurisation were higher for CH (over 1 Log·CFU g−1) than for RF (over 0.5 Log·CFU g−1) salmorejo and were not affected by storage time. Mesophilic spore counts (Log·CFU g−1) were similar in CH (2.51 Log·CFU g−1) and RF (2.98 Log·CFU g−1) untreated salmorejo. Pasteurisation allowed a higher Log reduction in mesophilic spores in CH (0.35–1.00 Log·CFU g−1) than in RF (0.05–0.75 Log·CFU g−1) salmorejo with some oscillations over time, although with a similar trend. Chill storage clearly inhibited the proliferation of mesophilic microorganisms in pasteurised salmorejo. In contrast, pasteurised salmorejo (either CH or RF) showed some enzyme activities during its shelf life (Pasteurisation treatments (either CH or RF) largely ensured the microbiological quality of salmorejo kept at 4 °C for 5 months . Initialelf life . PG was −1), used as a general oxidation index, were similar at the first month in the CH (19.6 mg 100 mL−1) and RF salmorejo (21.5 mg 100 mL−1), and then practically disappeared (<1.3 mg 100 mL−1). TBARS values, used as the lipid oxidation index, increased at the first month (>0.57 mg MDA kg−1) and then tended to decrease (up to 0.45 mg MDA kg−1) during the rest of storage. Both ascorbic acid and TBARS values confirmed that pasteurised salmorejo oxidized to a greater extent at the beginning of chill storage, coinciding with the period of higher activity of oxidase enzymes. The chromatic changes over time were modest. L* values remained quite stable over 59 CIE units during storage, with no clear signs of darkening. In contrast, the H* angle increased at the second month (from 52 to 66 CIE units), indicating the development of brown tones, and then remained stable. This succeeded because b* values increased while a* values decreased. ΔE values  occurred at the end of storage . The appL. monocytogenes, the criterion of 0.5 Log is considered appropriate in the European Union, equivalent to twice the estimated standard deviation (0.25) associated with the plate colony count  founf low pH ,19. SpecCloud clarification related to pectolytic enzymes is often considered a deterioration sign in many stored fruit juices. PME and PG are involved in the breakdown of pectins ; PME catUnlike stored fruit juices which can lose their natural turbidity, salmorejo is a tomato–oil thickened emulsion whose turbidity cannot be measured with a nephelometer (>4000 NTU), so a clarification trend was objectively assessed through flow behaviour (apparent viscosity) and texture parameters (deformation energy and hardness). This information was contrasted with the sensory assessment of consistency. As observed, a decrease in apparent viscosity was recorded at the beginning of storage, while texture changes over time were barely relevant. Sensory signs of clarification trend (lesser consistency assessed with mouth and spoon) were perceived at the end of storage. From a sensory viewpoint, early clarification does not appear to limit the shelf life of salmorejo, a thickened product, though the product turned less consistent as storage time and oxidation advanced, which contributed, together with other sensory alterations, to overall sensory deterioration. In gazpacho, a product less consistent than salmorejo, small changes in apparent viscosity and flow behaviour indexes during shelf life (30 days) could not be detected in the sensory assessment .2O2 as a catalyst for the oxidation of phenolic compounds and other substrates related to undesirable changes in flavour, texture, and colour [Oxidase enzymes also contribute to the deterioration of tomato products during shelf life. PPO catalyses the oxidation of the functional OH group attached to the carbon atom of the benzene ring of monohydroxy phenols to O-dihydroxy phenols and the dehydrogenation of O-dihydroxy phenols to O-quinones, which are highly reactive compounds that polymerize rapidly to form brown pigments that cause enzymatic browning . POD beld colour . Tomato d colour , while td colour . Reprodud colour , HTST atd colour  found thd colour  also repd colour . The oxi−1 in pasteurised gazpacho (90 °C for 5 min) kept at 4 °C for 60 days. MDA levels increased at the first month, suggesting the greater formation of secondary oxidised lipids  responsible for rancidity and other flavour alterations. Peroxidation reactions catalysed by POD enzymes might have enhanced lipid secondary oxidation. However, MDA levels were quantitatively low in pasteurised salmorejo, which did not develop rancidity during its shelf life. Fresh odour/flavour predominated over cooked odour/flavour (not included in QDA) as the product was heated for a few seconds at mild temperature. The freshness intensity was low compared to the untreated product (7.1), remained stable during most of storage, and likely decreased due to the accumulation of oxidized volatiles. Salmorejo has a complex flavour chemistry, involving many non-oxidized and oxidized volatiles from tomato, vinegar, garlic, olive oil, and other ingredients generated during processing and further storage. Liu et al. (2022) [Oxidative changes in salmorejo corresponded with the oxidase activities recorded. Browning was detected with the reflectometer (CIELab colour) earlier than with the sensory assessment. The rapid degradation of L-ascorbic acid, a reliable oxidation index used in fruit products , confirm. (2022)  studied . (2022) , brownin. (2022) , or the HPDE has been proven to be a suitable material for packing salmorejo. The bottle used is translucent and allows the product to be viewed. The appearance of salmorejo, in particular the colour and the separation of emulsion phases, can be examined by consumers before purchasing the product. This is not possible with Tetra Brik or opaque plastic containers. HDPE is stiff, strong, tough, chemical- and moisture-resistant, gas-permeable, and both easy to process and form. HDPE has a high crystallinity level and is less flexible, less transparent, but more resistant to heating than low-density polypropylene predominately used in film applications and in applications where heat sealing is necessary . For theThe shelf life of pasteurised–chilled salmorejo would be limited by a probable loss of its acceptance. Consumer acceptance towards “fresh-like” gazpacho has been studied by Fernández-Ruiz et al. (2017) . This prThe changes which occurred in salmorejo during its shelf life can be summarised as follows . The mosThe pasteurised–chilled salmorejo packed in HDPE bottles reached a long shelf life (4 months) compared to that of pasteurised tomato juices or purees. HTST pasteurisation (80 °C for around 10 s) by either conventional or radiofrequency heating can be used to obtain “fresh-like” salmorejo. Product stability is similar when operating conditions are adjusted in both pasteurizers to obtain similar levels of microbiological inactivation. Mesophilic and pathogenic bacteria are easily inhibited by the thermal treatment and when they further undergo chill storage in this acidic product. However, pasteurisation does not prevent salmorejo from showing some enzyme oxidation by PPO and POD at the beginning of its shelf life. Less intense thermal treatments than those referenced until now are required to obtain a stable salmorejo of good sensory quality. The shelf life of pasteurised–chilled salmorejo would be limited by long-term oxidative deterioration and their sensory implications. Future studies will elucidate how storage time affects consumer acceptance of this product. Economic costs and the energy efficiency of radiofrequency technology should be evaluated for its possible implementation in the food industry.


Ediacaran time (635–543 million years ago [Ma]) represents the dawn of macroscopic life following two Neoproterozoic Snowball Earth episodes , where aet al. [et al. could see that Ediacaran glaciation had occurred at least twice, not once, separated by as much as 20 million years.To answer these questions, we must know the style of glaciation. In a paper published in this Issue, Wang et al.  analysedet al. , thus eset al. . Consideen masse due to true polar wander (TPW) [They then devised a clever way of predicting ages on the ∼33 other glacial deposits using two simple rules. The first was to assume that there were no Ediacaran ‘Snowball’ glaciations, so the deposits would only have formed at mid-to-high latitudes. The second rule was that the continents had rotated largely er (TPW) ,10. TPW er (TPW) ,11,12, aer (TPW) . Alfred et al.’s [Wang et al.’s  resultinNumerous other tests of this novel hypothesis are also possible, such as the fact that sea-level rises should happen at localities that move towards the equator but those approaching the poles should experience sea-level drops . However


Glycine max [Linn.] Merr.) is a major challenge as there is little available information regarding non-parametric statistical origin approaches for Chinese domestic and imported soybeans. Commercially procured soybean samples from China (n = 33) and soybeans imported from Brazil (n = 90), the United States of America (n = 6), and Argentina (n = 27) were collected to characterize different producing origins using stable isotopes , non-metallic element content , and 23 mineral elements. Chemometric techniques such as principal component analysis (PCA), linear discriminant analysis (LDA), and BP–artificial neural network (BP-ANN) were applied to classify each origin profile. The feasibility of stable isotopes and elemental analysis combined with chemometrics as a discrimination tool to determine the geographical origin of soybeans was evaluated, and origin traceability models were developed. A PCA model indicated that origin discriminant separation was possible between the four soybean origins. Soybean mineral element content was found to be more indicative of origin than stable isotopes or non-metallic element contents. A comparison of two chemometric discriminant models, LDA and BP-ANN, showed both achieved an overall accuracy of 100% for testing and training sets when using a combined isotope and elemental approach. Our findings elucidate the importance of a combined approach in developing a reliable origin labeling method for domestic and imported soybeans in China.Verifying the geographical origin of soybeans ( Glycine max [Linn.] Merr.) evolved from wild soybean (G. soja Sieb. & Zucc.) and originated in China around 5000 years ago [Cultivated soybean  of plants reflect their growing environment, climate, and human interactions. Different photosynthetic pathways of plants and their cultivars have different biological effects on carbon dioxide respiration processes and uptake. These differences in plant tissue 13C/12C ratios are the main determinant of bulk δ13C signatures [15N values of naturally growing plants are mainly related to plant organs, plant age, climatic conditions, biological N2 fixation, and soil properties, and they are also susceptible to nitrogen fertilization [34S values are influenced by soil elements , atmospheric deposition , and sea spray in some coastal regions [δ34S values include soil sulfate reduction by anaerobic bacteria on a range of diffuse sources [Methods used to determine food geographical origins, such as stable isotopes and elemental content analysis, have been recognized as particularly useful origin discriminators as the elemental composition and stable isotope signatures  to track the origin of Ruditapes philippinarum (clam) shells, and the results showed shell ƐNd is a powerful tool to discriminate the geographic location of clams. Studies have reported the application of terahertz (THz) spectroscopy [Research endeavors have been conducted to ascertain the provenance and traceability of agricultural products using stable isotopes and elements combined with chemometrics: Cassiae Semen tea , Chinesetroscopy , energy troscopy , multi-etroscopy , and Chitroscopy . The geotroscopy ,35, and troscopy .δ2H, δ18O, δ15N, δ13C, and δ34S), non-metallic element content  and elemental profiles. The fingerprint information acquired by Isotope Ratio Mass Spectrometry (IRMS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) were combined with multivariate statistical techniques such as PCA, LDA, and ANN to distinguish the geographical origin of four origins, including domestic and imported soybeans. We identified individual isotope trends and undertook multi-variate analyses to establish the most useful discriminatory chemometric models for country of origin verification. This study proposes a combined approach to trace the geographical origin of soybeans in order to solve the phenomenon of mislabeled countries of origin in Chinese markets and improve traceability efficiency in the event of fraud or food safety concerns.To date, there are no studies that have generated an isotope or elemental database suitable for verifying the geographical origin of domestic and imported soybeans into China. The objective of this study was to examine the chemometric discrimination ability for the geographical origin of soybeans using stable isotopes (n = 90), China (n = 33), USA (n = 6), and Argentina (n = 27). Soybean samples were dried to facilitate storage and transportation. The specific sampling information is shown in All soybean samples were collected in 2020 from ports such as Shenzhen Port, Zhangjiakou Port, and others that are primarily involved in importing to China and analyzed by Shenzhen Customs, which resulted in the collection of 156 soybean samples from 4 countries: Brazil   equipped with an Elemental Analyzer (HT2000) , Germany), according to previously described methods [ methods ,37.δ15N, δ13C, δ34S and N, C, S content using a micro-balance  and tightly wrapped. The powdered samples were placed in an autosampler and converted to gases  in the EA. Evolved gases were passed through a drying tube containing Mg(ClO4)2. The EA combustion parameters were as follows: reaction oven: 1020 °C, column temperature: 70 °C, O2 flow: 175 mL/min, oxygen injection time: 3 s; carrier flow (helium gas): 200 mL/min.Around 3 mg of each powdered soybean sample was weighed in duplicate into a 4 × 6 mm tin capsule for the simultaneous determination of δ2H and δ18O. Soybean samples and reference materials were exposed to local atmospheric conditions to equilibrate exchangeable water in the laboratory for at least 48 h before analysis. TC/EA parameters were as follows: furnace temperature: 1350 °C, column temperature: 90 °C, carrier flow (Helium gas): 110 mL/min.Around 0.2 mg of each powdered soybean sample was weighed in triplicate into a 3.5 × 5 mm silver capsule for the simultaneous determination of δ) in accordance with the following formula:i and j denote the highest and the lowest atomic mass number of element E, respectively. PR and RefR represent the ratio of the heavier and the lighter element  of the sample and reference materials, respectively [The isotope ratio is denoted in delta notation , δ15N to the air (air) scale, δ34S to Vienna-Canyon Diablo Troilite (V-CDT) scale, while δ2H and δ18O were expressed relative to the Vienna-Standard Mean Ocean Water (V-SMOW) scale. The isotope values of soybean samples were calculated against international standard reference materials as follows: USGS43  was used for δ13C, δ15N and δ34S calibration, Sulfadiazine  was used as the quality control sample for elemental content of carbon, nitrogen, and sulfur . USGS43 , USGS54 , USGS56  were used for the δ2H and δ18O calibrations. Blanks were used, and standards were inserted for drift correction after every eight samples. The accuracy of carbon, nitrogen, and sulfur stable isotopes is ±0.1‰, ±0.2‰, and ±0.4‰, respectively. The accuracy of hydrogen and oxygen stable isotopes was ±1.2‰ and ±0.3‰, respectively.The 3  and 2.0 mL of H2O2  for 2 h. The digestion temperature program was as follows: from 0 °C to 100 °C in 10 min, increasing to 180 °C in 15 min, and increasing to 240 °C in 15 min before being kept at 240 °C for 10 min. After mineralization, the obtained extracts were redissolved to a total volume of 25 mL with double Milli-Q water (MΩ > 18.2 M) and subsequently analyzed by ICP-MS. A multi-element standard solution  in 0.5% nitric acid  was used to determine the sensitivity factors of all elements over the entire mass range measured by semi-quantitative mode dilution samples.A total of 23 macro, micro, and trace elements  were quantified using Inductively Coupled Plasma Mass Spectrometry . Around 0.5 g of powdered soybean was weighed and digested using a microwave digestion system. Samples were digested in Teflon digestion vessels containing a mixture of 6.0 mL of HNOp-value ≤ 0.05. Principal component analysis (PCA) was used in the analysis process to reduce the dimensionality of the dataset and describe the variability of the system with fewer variables. Linear discriminant analysis (LDA) was used to find the linear combinations of noted attributes that can clearly separate two or more classes of objects [Data processing was accomplished using SPSS 24.0 for macOS , and the results were expressed as the mean value ± standard deviation. GraphPad Prism 8.4.0 for macOS  was used to produce the figures. One-way analysis of variance (ANOVA) followed by the post hoc Tukey’s HSD was used to evaluate the differences in the samples with the isotope and elemental variations from the four countries, respectively. This study considered a statistically significant difference occurred at a  objects . Backprop < 0.05) among eight variables from the four origins . The δ2H, δ18O, δ15N, δ13C, and δ34S values ranged from −158.4‰ to −90.6‰, 12.8‰ to 27.9‰, −1.5‰ to 8.6‰, −30.0‰ to −26.0‰, and −8.7‰ to 8.1‰, respectively. Average δ2H, δ18O, δ15N, δ13C, and δ34S values were −121.9‰, 21.0‰, 0.7‰, −27.2‰, and 5.4‰, respectively. Stable isotope and elemental profiles of imported and Chinese domestic soybean samples were analyzed and summarized by mean value ± standard deviation in δ13C values were within the expected range for C3 plants, as δ13C values of all soybean samples ranged from −30.0‰ to −26.0‰. Mean δ13C values of soybean samples from the USA (−26.2 ± 0.3‰) were slightly more positive than other origins, and δ13C values of soybean samples from Argentina had the most negative mean δ13C values (−27.6 ± 0.6‰). Some environmental factors can affect the δ13C values of plants, such as drought, solar radiation intensity, low temperature, low pressure, and ozone stress [13C values of plants in humid cultivation areas were more negative [13C.Soybean e stress . Previounegative . Argenti15N values of Chinese soybeans (1.6 ± 1.7‰) was consistent with previously reported results [δ15N value of soybeans was 2.2 ± 0.2‰ from the USA, and the minimum mean δ15N value was found in Brazilian and Argentinian soybeans with an average of 0.4‰ and 0.5%. The use of nitrogen fertilizer is generally not recommended for legumes because, under favorable conditions, legumes are able to grow well from existing soil N plus N2 derived from symbiotic nitrogen fixation [δ15N value of soybeans from Brazil and Argentina is close to that of atmospheric N2 (0‰ by definition), and it is interpreted that these values result from the fractionation of the legume–Rhizobium symbiosis during biological nitrogen fixation (BNF), which is generally small. This results in the δ15N value of the legume approaching zero when there is high symbiotic dependence. [δ15N values of soybeans of the four studied origins. δ15N values typically rely on agricultural practices, such as the type of fertilizer applied (organic or synthetic) or agricultural management. Soybeans cultivated in China exhibit the largest δ15N standard deviation, potentially due to the different cultivation methods across China. It is possible that certain Chinese soybean samples have been cultivated using organic farming instead of conventional practices. Nonetheless, this hypothesis could not be substantiated due to the unavailability of soil and fertilizer data.The average of δ results ,41. The fixation . In ordeendence. ,43. Moreendence. . The natδ34S values of plants do not clearly distinguish production methods  or fertilizer type but are generally correlated with local geological and soil conditions [δ34S values (6.1‰ and 5.9‰), while the USA had the lowest mean δ34S values (−3.0‰). The δ34S values of USA soybeans had the widest isotopic distribution range compared to other origins, possibly due to a wider range of different geological sulfur sources and cultivation environments found in the Midwestern states of Iowa, Illinois, Minnesota, and Indiana (order of top producing states) [nditions ,45. Chin states) .δ2H and δ18O values were significantly different among the four origins. Argentinean soybean had the most positive δ2H and δ18O values . The lowest mean soybean δ2H value was found in the USA (−145.8‰), and the lowest mean soybean δ18O value was found in China (16.7‰). The climate of Argentina’s agricultural regions is more tropical (humid and warm) than other countries. In general, geographical temperature fractionation effects exist, where the concentration of 18O decreases with increasing latitude and cooler temperatures [δ2H and δ18O values for Chinese soybeans suggest they are grown inland and potentially in cooler regions than the other countries. A comparison between δ2H and δ18O values in soybean samples from four origins is shown in δ2H and δ18O values of soybeans show a reasonable overall linear relationship (R2 = 0.512), although soybean isotope values cluster into their respective origins. Soybean eratures . Howeverp < 0.05). USA soybeans had the lowest N, C, and S element contents among the four producing countries, while Chinese soybeans had the highest elemental contents. Some soybean samples from the USA may have originated from the northern states, which have cooler temperatures and less sunshine, resulting in lower nitrogen, carbon, and sulfur storage in plant tissues. Significant elemental differences in soybeans found in the four origins indicate that these elemental contents may contribute towards distinguishing geographical origin.Elemental nitrogen, carbon, and sulfur  of imported and domestic soybean samples ranged from 5.2% to 8.7%, 45.8% to 54.3%, and 0.1% to 1.0%, respectively. Average % N, % C, and % S contents were 6.2%, 50.4%, and 0.3%, respectively. A significant difference in N, C, and S elemental compositions of soybean from four origins was seen  linked to the geographical origin of the samples were found for all 23 elements except Mg, Fe, Cu, and Pb. Soybean Mg and Cu contents were found to be effective origin indicators of Chinese and Japanese soybeans [Mineral element abundance is shown in soybeans , but theElemental contents of Al, V, Co, Ga, Rb, Cs, and Tl were significantly higher in Brazilian soybeans than in the other three origins. Brazilian soybeans had the lowest concentrations of K, and Brazilian and Chinese soybeans had lower concentrations of AS, Se, and Cd than USA and Argentinean soybeans. Brazilian and Argentinean soybeans had lower concentrations of Ni. Chinese soybeans had significantly higher concentrations of Ni and the lowest concentration of Na. The elemental contents of Cr, Mn, Zn, and Se were significantly higher in USA soybeans than in other origins. Soybeans cultivated in Argentina had the highest concentrations of Na, As, and Sr and the lowest concentrations of Mn. Significant differences in mineral element contents are attributed to different geological and environmental conditions, climatic uptake factors, and human interventions from each origin. Soybean production in Brazil is concentrated in the “Cerrado” region, a prairie-like plain with less fertile soils that are rich in aluminum, highly acidic, and lack phosphorus and nitrogen . The lowChemometrics is an interdisciplinary method that employs multivariate statistics to extract essential information from intricate chemical systems and can now discern subtle differences in measurements among target products . In thisUnsupervised PCA was performed using a combination of stable isotopes and non-metallic and mineral element contents. Overall, PCA confirmed the ability of isotopes and elements to provide unsupervised origin classification and discriminate the soybeans based on origin. The first six principal components (PCs) in the PCA model accounted for 80.49% of the total variance of the original variables, and the first three PCs explained 58.05% of the total variance of the original variables. Meanwhile, a 3D-Ordinations plot, constructed using the first three PCs, showed that soybean samples from four origins were generally well clustered without overlap a. PCA waMore meaningful classification models were constructed using LDA and BP-ANN in combination with stable isotope and element data. In order to establish a stronger origin classification model, identify effective classification variables, and better classify soybean samples from different origins, supervised linear discriminant analysis (LDA) and unsupervised BP artificial neural network analysis (BP-ANN) were performed using both stable isotopes and multi-element contents.LDA analysis of soybean samples from four origins revealed discriminant accuracies of 85.3% for stable isotopes only, 75.0% for non-metallic elemental contents only, and 100.0% for mineral elemental compositions only  A–C. TheA BP-ANN model was built with 70% training samples and 30% testing samples. Training and testing samples from each origin were selected randomly. The normalized importance for each elemental and stable isotope variable for the contribution to origin verification is shown in While successfully differentiating soybeans from four origins using stable isotopes and mineral elements combined with chemometrics, it is crucial to acknowledge that bulk stable isotope values and mineral elements represent overall averages of components. In specific cases, these values may not fully capture isotopic and elemental variations among different samples. Recently, there has been increasing attention paid to Compound-Specific Stable Isotope Analysis (CSIA) due to its ability to provide unique information about food components at the molecular level, reflecting geographical, cultivation, and natural ecosystem processes . Our forδ2H, δ18O, δ15N, δ13C, and δ34S), non-metallic element contents  and 23 mineral elements to verify their geographical origins. PCA results showed the four groups of soybean samples were distributed independently. A combined stable isotope, non-metallic, and mineral element contents data fusion approach combined with LDA afforded a more accurate soybean origin classification (with discriminant accuracy rates of 100% for both the testing and training sets) than a single analytical approach. Likewise, BP-ANN gave a 100% accuracy rate for both testing and training sets of four origin groups, and As, Cd, Se, % S, and Fe were identified as the top five important variables. An accurate origin identification tool using isotope and elemental fingerprints combined with chemometrics could avoid any potential conflict of interest arising from incorrect origin identification among soybeans. Regulatory uptake and use of soybean geographical origin models will prevent fraud or mislabeling at a global market level and could contribute to fairer international trading.Stable isotope and elemental compositions were used to develop an origin identification model to determine the geographical origin of soybeans from four countries. The model could be further used to improve the origin traceability of similar leguminous food products. Chinese soybeans and imported soybeans were characterized by chemometric exploratory techniques and classification parameters such as PCA, LDA, and ANN using stable isotopes (


The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε–P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a  Hepatitis B virus (HBV) is a member of the Hepadnaviral family and is the smallest DNA virus known to infect animals. The HBV genome is 3.2 kilobases (kB) ,2,3,4,5,cis-acting RNA regulatory stem-loop known as epsilon (ε), situated at the 3′- and 5′-ends of the pregenomic RNA (pgRNA) ,4948,49]ynthesis ,53,54. Iynthesis .Regardless of the validity of these models, the translocation process must transform the ε–P complex. Initially, P facilitates protein priming with Y63 within the TP domain, then it enables DNA copying, and finally it replaces the ε template with DR1. Consequently, P must have distinct initiation and elongation modes, akin to protein-priming polymerases . The proTo ensure the formation of rcDNA and not merely the production of double-stranded and linear DNA, the RNA primer must be transferred to the 3′-proximal DR2 motif C. This scis-acting elements have been hypothesized to play important roles . . 123]. HAn alternative strategy that would circumvent the cccDNA-related issues would be to prevent the rcDNA from reaching the nucleus altogether. Given that nuclear transport depends on the nucleocapsid, this outcome can be achieved by capsid-targeting drugs, which are typically referred to as capsid assembly modulators (CAMs) and have been the topic of extensive review . In addiRather than eliminating the cccDNA, an alternative therapeutic strategy is to leverage the cell’s epigenetic machinery to functionally inactivate the cccDNA. One validated method to achieve this end is with IFN-α treatment ,136. In Interestingly, the HBV X protein was also found to be recruited to the cccDNA , suggestSince the ε–P interaction initiates protein-primed reverse transcription ,24,25,26Another approach to target the ε–P interaction is the Systematic Evolution of Ligands by EXponential Enrichment (SELEX) method to select strong P-binding RNAs that compete with ε for P binding . These “An alternative target of emerging anti-HBV therapies is protein priming, which offers the ability to prevent HBV replication at a very early stage. P is presumed to undergo a conformational change to transition from protein priming to the subsequent (−)-DNA strand elongation. This characteristic provides the opportunity to design anti-HBV therapeutic to specifically inhibit TP-mediated protein priming functions, complementing current NRTI treatments ,147,148.N-Hydroxyisoquinolinediones (HID), N-Hydroxynapthyridinones (HNO), N-Hydroxypyridinediones (HPD), and α-Hydroxytropolones (αHT) -DNA strand A. Conseqes (αHT) B. ImportOne major factor of HBV persistence is a defective immune response, including the depletion of cytotoxic T cells, lack of CD4+ T cell help, and the failure to produce neutralizing antibodies ,163,164.The most straightforward approach to rid the liver of cHBV infection is active elimination of existing cccDNA. One strategy to achieve this outcome is to mimic the immune-mediated clearance of cccDNA during acute HBV infection ,167. TheAn alternative approach to cccDNA elimination is genome editing with designer nucleases . This stGiven that NRTI and IFN-α therapies are not curative and require lifelong treatment ,16,17,18The secondary structure of a 61 nt ε has been determined A 21,22],2221,22]Fortunately, the solution NMR-derived structure of the truncated 27 nt ε upper helix (UH) ,182 has In conjunction with structural data, previous NMR studies have also assessed the dynamics of AL ε . SpecifiCurrently, there are no structures of HBV or Hepadnaviral P proteins, although homology models have been proposed for the RT  and RH  domain. Despite the limited structural understanding of P, an extensive body of knowledge exists about HFs that modulate HBV and Hepadnaviral P protein function. The first HF demonstrated to interact with P is the Hsp90 complex, which includes Hsp90, Hsp70, Hsp40, Hop/p60, and p23 ,140,141.The fundamental prerequisites for the ε–P interaction ,177, P–pIn the context of the pgRNA, an essential proximity between the 5′-end ε and the 5′-cap is required for efficient reverse transcription . MoreoveWhile these findings offer valuable insights, the biochemical methods are opaque to quantifying dynamic interactions and processes. Moreover, the mutational work may lead to unintended perturbations outside the regions being probed. Consequently, achieving a comprehensive mechanistic understanding of the ε–P interaction and its subsequent functions necessitates building on these findings with high-resolution structural dynamics studies. As previously mentioned, such data are entirely absent for P but have recently become available for full-length ε ,198.Our group recently determined the structure of full-length ε using a combination of small-angle X-ray scattering (SAXS) and solution NMR spectroscopy A 28]. O. O28]. OInterestingly, in vitro biochemical experiments suggest that nucleotides C14–G16 likely have a role in stalling the HBV replication complex, which potentially acts as a prerequisite for (−)-DNA strand transfer . AlthougAlongside structural measurements, our preliminary NMR dynamics data suggest that nucleotides in the PL (C14–C19 and adjacent U49), PTL (U32–U34 and C36), and U43 bulge undergo ps–ns motions C 28], a, a28], aTo begin evaluating the relevance of RNA dynamics for the ε–P interaction and its downstream functions, we combined solution NMR with molecular dynamics (MD) simulations to probe motions on the ps–ns, ns–1 μs, and μs–ms timescales. In agreement with our recent work C 28] an an28] anTogether, these data outline a series of complex motions on multiple timescales within the PL, PTL, and U43 bulge D. MoreovThe diversity of RNA sequence- and structure-specific requirements for HBV replication B suggestWithin this context, we posit that dynamics may correlate to function as follows. Considering that the PL and U43 bulge are required for P binding B 146,17, nucleotIn conclusion, our combined NMR and MD data reveal a series of complex motions on multiple timescales within full-length ε C,D. ThisSmall molecules present an opportunity to target RNA motifs such as pseudoknots, bulges, and hairpins, which are often highly conserved and mediate important biological functions ,206,207.As an initial step in testing our structure-informed hypothesis, we employed a small molecule microarray (SMM) approach, which has been previously employed to identify various chemotypes targeting RNA ,212,213 Subsequently, NMR titration experiments were employed to distinguish specific binders from nonbinders, aggregators, and nonspecific binders. This analysis revealed that Raloxifene selectively targets the ε PL . Raloxif50) of approximately 70 μM and 110 μM, respectively, whereas Arzoxifene does not bind  ofTo gain further insight into the ε–Raloxifene interaction, computational docking and MD simulations were conducted. Raloxifene docking pose predictions indicated that among the top-10 NMR ε conformers , three c3 (SG74) severely reduced affinity -DNA strand by phosphorimaging. Unfortunately, neither Raloxifene nor SG92 affected HBV protein priming . . 219]. IInterestingly, both Raloxifene and Daclatasvir have been shown to regulate the dynamics of the ε PL. As discussed in Approximately two billion people worldwide have been exposed to HBV. This global exposure has resulted in over 300 million cHBV infections and around 800,000 deaths annually ,64,65, wHowever, the lack of structural information for the ε–P complex and P itself prevents structure-informed design of anti-HBV therapeutics. Our recent structure of full-length ε  Figure A is a crOur first approach involved an HTS-based approach . We screFuture experimental work in this area must focus on determining high-resolution structures of ε–small molecule complexes and employing structure–activity relationship studies to reveal chemotypes that selectively target the ε PL. Given the inherent dynamics of full-length ε ,198, com


Smoking and vaping are linked to lung inflammation and lowered immune response.Examine the prevalence of coronavirus disease 2019 (COVID‐19) cases, testing, symptoms, and vaccine uptake, and associations with tobacco product use.Data came from the 2021 National Health Interview Survey. The 2021 Sample Adult component included 29,482 participants with a response rate of 50.9%. We investigated COVID‐19‐related outcomes by tobacco product use status and reported national estimates. Multivariable regression models were performed accounting for demographics , serious psychological distress, disability, and chronic health conditions.In our regression analyses, odds of self‐reported COVID‐19 infection were significantly lower for combustible tobacco product users . Combustible tobacco users also were less likely to report ever testing for COVID‐19 , ever testing positive for COVID‐19 , and ever receiving COVID‐19 vaccine  compared with their nonuser peers. Compared to nonusers, users of any type of tobacco who contracted COVID‐19 had higher odds of losing smell , which was more pronounced among exclusive e‐cigarette users. The odds of receiving vaccine were lower for all current exclusive tobacco product users compared to nonusers (AORs = 0.40 to 0.70).Continued monitoring of tobacco product use and its association with respiratory diseases such as COVID‐19 is crucial to inform public health policies and programs. In addition, efforts to promote vaccination, especially among tobacco product users, are warranted. What's known: Smoking and vaping are linked to lung inflammation and can lower immune response. What's new: Combustible tobacco users had lower odds of self‐reported coronavirus disease 2019 (COVID‐19) infection, testing, and vaccination compared with nonusers. Tobacco users who got COVID‐19 had higher odds of losing smell, especially exclusive e‐cigarette users. All current exclusive tobacco product users were less likely to get vaccinated compared with nonusers. Clinical implications: Continued monitoring of tobacco use and COVID‐19 is needed to inform public health policies. Results highlight the importance of promoting COVID‐19 vaccination among tobacco users. Quitting smoking and vaping could reduce COVID‐19 risks. Findings suggest tobacco use is linked to lower testing and vaccine uptake as well as higher olfactory symptoms if infected. In brief, due to the COVID‐19 pandemic, typical data collection procedures were disrupted in 2021 NHIS. From January to April, household members were initially contacted via telephone, with subsequent personal visits authorized. This approach continued from the latter part of 2020. Starting in May 2021, interviewers returned to standard survey interviewing procedures, where initial contact with household members was attempted in person, followed by telephone if necessary. Interviewers were required to wear masks, practice social distancing, and be fully vaccinated if interviews were conducted in the home. In 2021, 62.8% of the sample adult interviews were conducted at least partially by telephone.The survey description and data collection methodology for the 2021 NHIS can be found at the Centers for Disease Control and Prevention website at 2.22.2.1being told having/tested for COVID‐19 [(1) “Has a doctor or other health professional ever told you that you had or likely had coronavirus or COVID‐19?” (2) “Have you ever been tested for coronavirus or COVID‐19?” (3) “Did the test find that you had coronavirus or COVID‐19?”], COVID‐19 symptoms . Symptoms questions were restricted to only participants who ever had COVID‐19. For vaccine uptake, participants were asked [“Have you had a COVID‐19 vaccination?”] and number of vaccines [“How many COVID‐19 vaccinations have you received?”]. All answers were yes versus no, except for the severity of symptoms (non/mild vs. medium/severe) and the number of vaccines received (≥2 vs. 1).Participants were asked a series of questions about COVID‐19, including 2.2.2Current cigarette smoking was defined as having smoked at least 100 cigarettes in one's lifetime and now smoking every day or some days. Current use/smoking of other products , cigars, pipes/hookah, or smokeless tobacco) was defined as ever using the product and now smoking or using it every day or some days. Any tobacco product use was defined as ever used and now using “every day” or “some days” at least one tobacco product. Any combustible tobacco product use was defined as using on “every day” or “some days” at least one combustible tobacco product: cigarettes; cigars, cigarillos, filtered little cigars; pipes, or hookahs. Further, we defined exclusive use of each product as currently using only one product .2.2.3Demographic variables included age groups , sex , sexual orientation , race/ethnicity (non_Hispanic (NH)‐White, NH‐Black, Hispanic, or Other race), education , poverty level , employed (no or yes), social distancing at work in the past 7 days (no or yes), region .Disability was defined based on the self‐reported presence of selected limitations, including vision, hearing, mobility, remembering, self‐care, and communication. Respondents answered six questions: (1) Do you have difficulty seeing, even when wearing glasses? (vision); (2) Do you have difficulty hearing, even when using a hearing aid? (hearing); (3) Do you have any difficulty walking or climbing steps? (mobility); (4) Using your usual language, do you have difficulty communicating, for example, understanding or being understood? (communication); (5) Do you have difficulty remembering or concentrating? (cognition); and (6) Do you have difficulty with self‐care, such as washing all over or dressing? (self‐care). Response items were “no difficulty,” “some difficulty,” “a lot of difficulty,” or “cannot do at all.” Respondents were categorized as having a disability if they reported “a lot of difficulty” or “cannot do” at all to any of the six questions mentioned earlier.The Kessler Psychological Distress Scale is a series of six questions that ask about feelings of sadness, nervousness, restlessness, worthlessness, and feeling like everything is an effort in the past 30 days. Participants responded on a Likert scale ranging from “None of the time” (score = 0) to “All of the time” (score = 4). Responses were summed over the six questions; individuals with a score of ≥13 were coded as having serious psychological distress, and respondents with a score of <13 were coded as not having serious psychological distress.Chronic health condition was collected by asking, “Ever been told you had coronary heart disease, high cholesterol, angina, heart attack, stroke, cancer,” or “ever had diabetes, asthma, weak/failing kidneys, weakened immune system due to prescriptions, epilepsy or having a current respiratory allergy.” Those who answered “no” to all questions were considered not to have chronic health conditions.2.3α = 0.05.Unweighted frequencies and weighted percentages with corresponding 95% confidence intervals (CI) of measures associated with COVID‐19  were calculated overall and by current tobacco product use and other covariates. To assess an association between each COVID‐19 measure with tobacco product use, multivariable models were performed with the COVID‐19 measure  as the outcomes and tobacco product use as the exploratory variable controlling for all other study variables  currently used any tobacco product, 14.2% (35.6 million) used any combustible tobacco product, and 3.3% (8.3 million) used two or more tobacco products  were ever told by a physician or health professional that they had COVID‐19, 58.8% (27 million) were ever tested for COVID‐19, and 10.9% (5 million) ever tested positive for COVID‐19. Among tobacco product users who provided information on the COVID‐19 vaccine (34.1 million), 57.3% (19.6 million) ever received the COVID‐19 vaccine, and 84.9% of them (16.6 million) received at least 2 doses of the vaccine.Among tobacco product users who reported contracting COVID‐19; 65.8% (3.9 million) and 61.7% (3.6 million) reported a loss of smell or taste, respectively.p‐values < 0.05). Those who used any type of tobacco products  and ENDS users  were more likely to report loss of smell due to COVID‐19. Compared with never users, current exclusive users of cigarettes , ENDS , cigars , and smokeless tobacco  were less likely to report ever receiving a COVID‐19 vaccine. Exclusive pipe/waterpipe users were more likely to have been tested for COVID‐19 .In multivariable regression models, compared to nonusers of tobacco products, those who used any combustible tobacco product were less likely to report COVID‐19 , to have ever been tested for COVID‐19 . Those who used two or more tobacco products were less likely to receive the COVID‐19 vaccine   have been explained.


SMN1) gene, and consequent loss of function of SMN protein, which results in progressive loss of lower motor neurons, and muscular wasting. Antisense oligonucleotide (ASO) nusinersen (Spinraza®) modulates the pre–mRNA splicing of the SMN2 gene, allowing rebalance of biologically active SMN. It is administered intrathecally via lumbar puncture after removing an equal amount of cerebrospinal fluid (CSF). Its effect was proven beneficial and approved since 2017 for SMA treatment. Given the direct effect of nusinersen on RNA metabolism, the aim of this project was to evaluate cell-free RNA (cfRNA) in CSF of SMA patients under ASOs treatment for biomarker discovery.Spinal muscular atrophy (SMA) is a rare autosomal-recessive neurodegenerative disorder caused by mutations in survival motor neuron 1 , and compared to baseline (T0).We observed the deregulation of cfRNAs in patients at T6 and we were able to classify these RNAs into disease specific, treatment specific and treatment dependent. Moreover, we subdivided patients into “homogeneous” and “heterogeneous” according to their gene expression pattern. The “heterogeneous” group showed peculiar activation of genes coding for ribosomal components, meaning that in these patients a different molecular effect of nusinersen is observable, even if this specific molecular response was not referable to a clinical pattern.This study provides preliminary insights into modulation of gene expression dependent on nusinersen treatment and lays the foundation for biomarkers discovery.The online version contains supplementary material available at 10.1186/s13062-023-00413-6. SMN1) gene (locus 5q11.2-q13.3), which lead to the loss of functions of the encoded protein SMN. However, up to 10% of full-length SMN is produced by the centromeric homologous SMN2 gene, which partially compensates the lack of SMN1. Hence, SMN2 copy number has been found inversely correlated with disease severity, contributing decisively to the observed SMA clinical heterogeneity [SMN2 gene, inducing the production of a greater amount of full-length, biologically active, SMN. Nusinersen shown to ameliorate motor function in pediatric [Spinal muscular atrophy (SMA) is a rare autosomal-recessive neurodegenerative disorder (incidence 1/8000 live births) characterized by progressive loss of lower motor neurons, with subsequent muscular atrophy and, in the most severe forms, early death due to respiratory failure. A wide range of SMA phenotypes has been described according to age of onset and level of motor functions achieved, ranging from in utero to adult onset SMA type 0–4) [ [SMN1) gediatric , and aduediatric . Nusinerediatric –11. Thesediatric . Both thA recent published work identified miRNAs as potential biomarkers to monitor response to nusinersen in pediatric SMA patients. This study demonstrated that muscle-specific miRNA serological levels decrease over disease course upon nusinersen treatment identifying these circulating molecules as possible non-invasive biomarkers of nusinersen therapeutic effect in SMA patients . In addiIn this work we will present a quantitative explorative study of mRNAs and lncRNAs analyzed by RNA-sequencing at baseline, i.e. before first injection of nusinersen (T0), and at fifth injection, after 6 months of nusinersen treatment (T6).A cohort of 18 genetically defined pediatric SMA type 2 and 3 patients followed-up at Child Neuropsychiatry Unit of IRCCS Mondino Foundation  and at the Developmental Neurology Unit of Fondazione IRCCS Istituto Neurologico Carlo Besta , was included in the study  and after 6 months of treatment (T6). Biological samples were stored at − 80 °C until use.g for 10 min at room temperature and immediately stored at − 80 °C. RNA was extracted from 400 µl of CSF with miRNeasy Serum/Plasma Kit  following manufacturer’s instructions. Then, RNA was quantified and its quality assessed with Nanodrop ND-100 spectrophotometer .After withdrawal, CSF samples were centrifuged at 2000 × RNAs to be sequenced were processed with RiboCop rRNA Depletion Kit  starting from 50 ng of RNA. Sequencing libraries were prepared using CORALL Total RNA-Seq Kit  following manufacturer’s instructions and performing 20 PCR cycles. Then 1.8 pM of pooled and denatured libraries were loaded on a NextSeq 500/550 High Output Kit v2.5 (150 Cycles) .Bioinformatic analysis for obtaining differentially expressed (DE) genes and gene enrichment analyses were conducted as in Garofalo et al. , except To identify cfRNAs in CSF associated with nusinersen treatment of SMA patients, we performed RNA-sequencing analysis through Next Generation Sequencing (NGS) approach. Eighteen CSF cfRNA samples obtained from all pediatric SMA patients  treated with nusinersen at T6 were compared to CSF cfRNA samples from the respective patients at T0. We detected 48 DE cfRNAs , we divided DE cfRNAs into three categories: 1) disease specific, mostly mRNAs already involved in pathways affected in SMA pathogenesis  , we performed an enrichment analysis of genes by Gene Ontology (GO) database. This analysis is useful for high-throughput data interpretation since it highlights biological phenomena that may be represented by DE genes . PracticWe queried Biological Process, Cellular Component and Molecular Function Fig. A–C. Fromn = 6) clustered differently from the whole group. Thus, we splitted the analysis by evaluating DE cfRNAs separately for the two subgroups of patients. We defined as “heterogeneous” those who appeared to be transcriptionally different from the T0  of all DE cfRNAs in SMA patients at T6 versus T0 Fig. . From thFurther, we conducted a gene database/literature search for evaluating whether the DE cfRNAs in these two subgroups were disease specific, treatment specific or treatment dependent. For the “heterogeneous”, we reported this classification in Fig. To show in which events were involved the DE cfRNAs we found in the “heterogeneous” group, we used GO database and showed enriched terms for Biological Process, Cellular Component and Molecular Function Fig. A–C. FromEventually, DE cfRNAs emerged from each analysis were compared. No cfRNAs were commonly DE in the three approaches. Two DE cfRNAs were common in the “all” and in the “heterogeneous” groups, while 5 DE cfRNAs were shared among the “all” and the “homogeneous” group , since in a case of type 0 SMA, negative myotubularin immunohistochemical staining was showed, suggesting the dependence of this family of proteins to SMN [SMN2 promoter activation [SMN2 expression reduction [SMN2 as we observed it up-regulated. Moreover, we postulated a link between HNRNPA1L2 and IARS2  that was classified as disease specific. Indeed, during mitochondrial stress, a feature of SMA molecular pathogenesis, tRNAs are released from the mitochondrial matrix to the cytosol, where they can interact with hnRNPA1 protein in the cytosol [We investigated modification of cfRNAs expression in CSF of type 2 and 3 pediatric SMA patients after five nusinersen injections , compared to first injection . Our aim was to observe how nusinersen affects gene expression and whether cfRNAs in CSF, which is removed before each treatment injection, may be addressed as prognostic biomarkers. We found 48 DE cfRNAs, mostly down-regulated mRNAs. After consulting gene databases (such as Ensembl and NCBI) and the literature (PubMed), we selected DE mRNAs directly associated with disease (disease specific), directly associated to nusinersen treatment (treatment specific) and those modulated in response to nusinersen treatment (treatment dependent). We addressed ARHGEF19, COX6C, MTMR14, TBL1XR1, KCNK2 and IARS2 as treatment specific ones. ARHGEF19 (Rho/Rac Guanine Nucleotide Exchange Factor 18) belongs to the Rho GTPase GEF family, that is implicated in regulation of axon formation, growth, guidance and branching , 18. Lowing gene . This cas to SMN . TBL1XR1tivation . KCNK2 . HSP90 inhibitors were indeed tested in a preclinical model of SMA showing amelioration of motor neuron degeneration, due to the enhanced proteasomal degradation of the Hsp90 client proteins .Further, all mRNAs within the “heterogeneous” group that we classified as treatment specific, RPL3, RPS8, RPL35A, RPL5, RPS27A and RPS4X, code for ribosomal subunits, demonstrating a strong correlation with nusinersen treatment, given the involvement of SMN in ribosome biology . Among tCorrelation analysis between RNA profile in the two groups and clinical data has been run but we found no associations. A limitation in this instance might be the relatively small number of patients to perform clinically meaningful stratifications. On the other hand, we correlated only clinical features that may be quantified, this approach excludes all clinical observations that are not captured by the functional motor scoring such as subjective motor improvement or response to fatigue. Also, we cannot exclude that clinical correlations may be found after a longer treatment follow-up. We hypothesized that the nusinersen effect is not yet evident in peripheral tissue as CSF after only 6 months. We plan to measure the identified markers at next infusion time point to check their expression. In particular, the spontaneous division of SMA patients in two groups may be understood first by increasing the number of samples to confirm this clusterization and also by increasing the time points may studies to figure out if this separation is maintained and if it is related to other clinical features.SMN2-targeting.Hence, our results point the way for more extensive investigations about cfRNAs role as biomarkers of nusinersen effect, and highlight the implication of the discussed molecular pathways in SMA pathogenesis, and their modulation after We identified specific cfRNAs that may be used to monitor the early effect of ASOs treatment at a molecular level. Moreover, we distinguished a group of patients, named “heterogeneous”, whose transcriptomic signature differed from the group of patients that uniformly clustered as the results of PCA analysis, and also when compared to the analysis where all patients were considered. By examining in depth, the “heterogeneous” group, we realized that DE cfRNAs were highly associable to nusinersen treatment. These observations made us speculate that this group is the most responsive to the treatment at molecular levels, despite transcriptomic observations were not clearly transferable to clinical levels, possibly because the small number of patients and the short-term evaluation. Thus, based on the current findings we aim to extend our investigation by testing CSF samples in a wider cohort and at successive injections to further characterize and corroborate the potential role of these molecules in SMA longitudinal assessment.Additional file 1. File excel composed by 4 sheets. Table S1. All DE. Differentially expressed cfRNAs in “all” SMA patients at T4 versus T0 nusinersen injection. Gene name, gene biotype and measured log2FC are reported for each transcript. Only transcripts with |log2(T4/T0l)|≥ 0.5 and a FDR ≤ 0.1 are shown. Table S2. Heterogeneous DE. Differentially expressed cfRNAs in “heterogeneous” SMA patients at T4 versus T0 nusinersen injection. Gene name, gene biotype and measured log2FC are reported for each transcript. Only transcripts with |log2(T4/T0l)|≥ 0.5 and a FDR ≤ 0.1 are sown. Table S3. Homogeneous DE. Differentially expressed cfRNAs in "homogeneous" SMA patients at T4 versus T0 nusinersen injection. Gene name, gene biotype and measured log2FC are reported for each transcript. Only transcripts with |log2(T4/T0l)|≥ 0.5 and a FDR ≤ 0.1 are shown. Table S4. Venn Results. Venn analysis of DE cfRNAs in “all”, “heterogeneous” and “homogeneous” groups.


This core–shell structure provides insight into a potentially unique nucleation and growth mechanism—nanocluster cores may nucleate through local, spinodal-like compositional fluctuations in Ni, with subsequent growth driven by rapid Si diffusion. This study underscores how APT can shed light on an unusual irradiation-induced nanocluster nucleation mechanism active in the ubiquitous class of austenitic stainless steels.Austenitic stainless steel D9 is a candidate for Generation IV nuclear reactor structural materials due to its enhanced irradiation tolerance and high-temperature creep strength compared to conventional 300-series stainless steels. But, like other austenitic steels, D9 is susceptible to irradiation-induced clustering of Ni and Si, the mechanism for which is not well understood. This study utilizes atom probe tomography (APT) to characterize the chemistry and morphology of Ni–Si nanoclusters in D9 following neutron or proton irradiation to doses ranging from 5–9 displacements per atom (dpa) and temperatures ranging from 430–683 °C. Nanoclusters form only after neutron irradiation and exhibit classical coarsening with increasing dose and temperature. The nanoclusters have Ni Austenitic stainless steels (SS) such as 304 SS and 316 SS are the most ubiquitous structural alloys in light water reactor (LWR) nuclear power plants . But the22 n/cm2) [The FFTF experiments focused on the irradiation-induced mechanical property evolution of D9. Over irradiation temperatures of 390–457 °C, fracture toughness degrades with increasing dose from ~45 dpa to ~135 dpa (corresponding to neutron fluences 9–27 × 102 n/cm2) . More ge2 n/cm2) . Meanwhi2 n/cm2) ,22 speci2 n/cm2) . This ki2 n/cm2) ,23. The Despite these aforementioned irradiation effects studies on alloy D9, the irradiated microstructure and mechanical properties had not been directly related until Chen and colleagues  conducteA unique aspect of the irradiated microstructure that remains to be fully explored is this Ni–Si nanoclustering. Although Chen et al.  characteThe present study accesses the identical neutron-irradiated specimens of D9 previously characterized by Chen et al. . The as-An ingot of Alloy D9 was obtained from Oak Ridge National Laboratory . The alloy composition is provided in 13 n/cm2-s (E > 1 MeV), during cycles 143A through 150B; the specimen loading configuration is described in ref. [22, 2.69 × 1022, and 3.07 × 1022 neutrons/cm2, corresponding to damage doses of 5, 8, and 9 displacements per atom (dpa), respectively. The irradiation temperatures were determined to be 448, 430, and 683 °C, respectively [Discs were loaded into capsules for neutron irradiation in the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL) . The capsules were irradiated in the ATR East Flux Trap, which has a maximum fast flux of ~9.7 × 10 in ref. . Three Dectively , based oectively  with temA proton irradiation was also conducted to conditions falling within the dose and temperature range bounded by the neutron irradiations, as shown in APT needles were fabricated using established focused ion beam (FIB) milling procedures on a Thermo Fisher Scientific Quanta 3D FEG dual-beam SEM/FIB  at the Center for Advanced Energy Studies (CAES) . APT neeThe needles were tested in a CAMECA  Local Electrode Atom Probe (LEAP) 5000X HR, also at CAES, operating in laser pulse mode. The LEAP run parameters for each needle are summarized in maxd, and the minimum number of ions that must satisfy the maxd condition to be considered a cluster, minN, were varied for each needle following recommendations from refs. [maxd and minN were consistent within each irradiation condition and varied slightly across irradiation conditions, as shown in GD, as [gR is the overall radius of gyration, which is estimated from the gyration radii in the x, y, and z directions for each cluster [Cluster analysis was conducted within IVAS using the maximum separation method  for Si iom refs. ,37,38,39n DG, as :(1)DG=25 cluster :(2)Rg=RgTV is the analyzed volume of a given needle, and CN is the number of clusters identified using the maximum separation method.The nanocluster number density was determined by:Three APT needles were reconstructed from the as-received material, and five from the proton-irradiated specimen. All of the reconstructions show homogeneous distributions of all major alloying elements, Fe, Cr, Mn, Ni, and Si, in both the as-received and proton-irradiated specimens, as shown in The proton-irradiated needles reveal no evidence of irradiation-induced nanoclustering or nanoprecipitation. It is also worth noting that no chemical segregation to features such as grain boundaries, dislocation lines, or dislocation loops is observed in any of the as-received or proton-irradiated needles. However, the APT needle preparation process did not specifically aim to capture grain boundaries. Additionally, the likelihood of capturing dislocation loops or lines in the nanoscopic volume of an APT needle is low, and such features can also pop out to the surface of needles upon application of the APT laser pulse. Hence, no conclusive statements about nano-segregation behaviors can be drawn.A total of six, two, and five APT needles were reconstructed from the 5, 8, and 9 dpa neutron-irradiated specimens, respectively, with representative reconstructions shown in 24 m−3 at 5 dpa, through to 1.31 ± 0.31 × 1024 m−3. This coarsening behavior is reflected by an increase in nanocluster volume fraction, which is 0.19, 0.21, and 0.45, at 5, 8, and 9 dpa, respectively. The volume fraction increase is only statistically significant between the 8 dpa, 430 °C and the 9 dpa, 683 °C conditions, suggesting that volume fraction increase may be most strongly influenced by temperature. The nanocluster size distribution, as shown in The average nanocluster morphologies are also summarized in 3Si stoichiometry). At the lower irradiation temperatures, Ni isoconcentration surfaces cannot be defined, confirming that the nanoclusters do not achieve Ni3Si stoichiometry. At the higher irradiation temperature, the isoconcentration surfaces for Ni are smaller than for Si, as shown in Nanoclusters appear to have a core–shell type structure in which the Ni-rich core is surrounded by a Si-rich shell, as shown in One APT needle from the 8 dpa, 430 °C neutron-irradiated specimen passed over a grain boundary. The RIS behavior across this boundary is summarized in Irradiation-induced Ni–Si nanoclusters or γ’-phase nanoprecipitates have long been known to form in austenitic stainless steels ,52,53,54Although the summarized data are limited and were collected over nonsystematic irradiation conditions and from different process heats of alloys, a few general trends can be extracted. First, the size and number density evolution of nanoclusters may suggest coarsening with increasing dose and/or temperature; this can be seen visually with bubble plots, as shown in 3Si stoichiometry [3Si stoichiometry , as shown in 3Si stoichiometry is the Fe-17Cr-12Ni-1Si model steel irradiated with 6.4 MeV Fe3+ ions at 200 °C, from Fukumoto et al. [3Si nanocluster stoichiometry.At elevated doses, Ni–Si nanoclusters are generally considered to be γ’-phase precipitates, which have simple cubic structures with Nihiometry ,56 and lhiometry . The curo et al. . They chThe Ni–Si precipitation mechanism remains unclear. Historically, Ni–Si precipitates have been thought to initially nucleate at interstitial sinks ,57, suggAlternatively, the Ni core–Si shell structure of the nanoclusters observed in the present work may be able to shed light on possible nucleation mechanisms. This core–shell structure has not been reported in other studies; although Toyama et al.  describe3 stoichiometry, which makes the hypothesized mechanism plausible. The nanoclusters are known to be coherent with the matrix, and their growth is believed to be promoted by coherency strain [In austenitic stainless steel or γ-Fe, Si  has a hiy strain . This, ty strain .3+ ion irradiation in a study from Fukumoto et al. [Finally, it is also worth commenting on the absence of Ni–Si nanoclusters in the proton-irradiated specimen herein. This observation is consistent with previous studies showing that ion ,74 and po et al. . Nevertho et al. .3Si  stoichiometry. The stability of these nanoclusters at the highest irradiation temperature, 682.5 °C, suggests that Ni–Si nanoclusters may be able to exist over a wider irradiation temperature range than previously thought. The observed nanoclusters exhibit a core–shell structure with a Ni-rich core and a more diffuse Si shell. Although a core–shell structure has not previously been reported for Ni–Si nanoclusters, it is consistent with post-irradiation annealing in the archival literature, which shows Si recovers more readily than Ni from nanoclusters. Because Ni is a slower diffuser than Si, the core–shell structure suggests that the Ni cores may nucleate through local compositional fluctuations akin to a spinodal-like phase instability, then subsequently grow through rapid Si diffusion to the core–matrix interface.APT was used to investigate austenitic stainless steel D9 irradiated with neutrons to 5 dpa at 448 °C, 8 dpa at 430 °C, and 9 dpa at 683 °C, and with 2 MeV protons to 7.0 dpa at 500 °C. Irradiation-induced nucleation of Ni–Si nanoclusters are consistently observed in all neutron-irradiated specimens, but they do not nucleate under proton irradiation. The nanoclusters coarsen with increasing neutron irradiation dose and temperature, but the Ni/Si ratio remains relatively constant near Ni


In the article titled “Development of Auer bodies from giant inclusions associated with rough endoplasmic reticulum in acute promyelocytic leukemia,” published on pages 111-117, Volume 5, Issue 2 of Blood Science, the figure caption for figure 6 has been changed for accuracy in the online version.“Figure 6. Mature Auer bodies. (a) and (b) show myeloperoxidase reactivity of promyeloblasts. (a) illustrates Auer bodies (arrowheads) – one of them irregular in shape – and granules in the perinuclear space (arrows), N, nucleus (unstained) ×10k; (b) shows longitudinal sections (arrows) and cross-sections (arrowheads) of Auer bodies in promyeloblasts, ×5k; (c), an Auer body has a double-crystalized center and an enveloping membrane (arrowhead) similar to that of the nuclear membrane (arrow), ×15 k; (d), an Auer body with a single crystallized center (arrows), ×15 k.In addition, figure 6 has been replaced with an updated version online.