However, it is problematic for other ceramic materials , especially porous ones. In this case, it is mainly due to poor adhesion to the substrate, resulting in strong degradation of the graphene. For these reasons, the research topic of this study was undertaken. This article presents research on the development of the methodology of graphene transfer onto ceramic Al2O3 surfaces. Polycrystalline graphene chemical vapour deposition (CVD) monolayer and quasimonocrystalline high-strength metallurgical graphene (HSMG®) synthesised on liquid copper were used. When developing the transfer methodology, the focus was on solving the problem of graphene adhesion to the surface of this type of ceramic, and thus reducing the degree of graphene deterioration at the stage of producing a ceramic–graphene composite, which stands in the way of its practical use. Plasma and chemical ceramic surface modification were applied to change its hydrophobicity, and thus to improve the adhesion between the graphene and ceramic. The modification included the use of dielectric barrier discharge (DBD) plasma, oxygen plasma , and hydrofluoric acid treatment. Changes in surface properties caused by the modifications were determined by measuring the contact angle and  measuring the degree of surface development. The effectiveness of the applied surface preparation methodology was evaluated based on the damage degree of CVD and HSMG® graphene layer transferred onto modified Al2O3 using optical microscopy and Raman spectroscopy. The best average ID/IG ratio for the transferred HSMG® graphene was obtained after oxygen plasma modification (0.63 ± 0.18) and for CVD, graphene DBD plasma was the most appropriate method (0.17 ± 0.09). The total area of graphene defects after transfer to Al2O3 was the smallest for HSMG® graphene after modification with O2 plasma (0.251 mm2/cm2), and for CVD graphene after surface modification with DBD plasma (0.083 mm2/cm2).Graphene transfer onto ceramics, like Si/SiO Graphene, thanks to its unique mechanical, thermal, and electrical properties, is widely known as an innovative material of the future ,2,3,4,5.®) [The methods of production of large-area graphene include synthesis on solid metal by chemical vapour deposition (CVD) , synthes®) ,12,13. AMany literature reports concern the use of graphene in filtration, but these are solutions based mainly on polymer membranes ,21,22,232) is used as the target ceramic substrate. Silicon itself or its oxide are widely used substrate materials for studying the effectiveness of improved transfer methods. The lack of use of other ceramic materials for this purpose is explained by their surface morphology and physicochemical properties, by which graphene shows poor adhesion to ceramics and is damaged during the removal of the supporting polymer [Most of the graphene transfer methods presented in the literature refer to procedures where quartz  groups on the surface. Commercial membranes are modified by hydrophobization for use in separation processes. This effect would also be possible by covering them with a layer of graphene ,36,37. O2 plasma  were pro2 plasma .® methods was used for the study. The surface of Al2O3 ceramics was modified by means of barrier plasma, oxygen plasma, and etching with hydrofluoric acid. The surface changes were assessed based on FTIR (Fourier Transform Infrared Spectroscopy) tests and the wetting angle measurement. The quality of the transferred graphene layers was evaluated based on optical microscopy and Raman spectroscopy. On this basis, the relationship between the state of the ceramic surface and the obtained quality of graphene transferred to its surface was demonstrated. We also determined which of the surface modification methods is optimal for obtaining the best quality transferred graphene layer.Bearing in mind, on the one hand, the potential benefits of using graphene–ceramic composite membranes, and on the other hand, the problems with transferring the graphene layer to ceramics, the purpose of this work were focused on this basis. The work aimed to determine the effect of chemical and physical modifications of the surface of the ceramic porous membrane on the quality of the transferred graphene. Large-area graphene produced by the CVD and HSMG2O3 discs with a diameter of 47 mm and pore size <0.1 µm  were used for surface modification and graphene transfer. CVD graphene synthesized on solid copper and HSMG® graphene synthesized on liquid copper were used for transfer onto ceramics.Porous oxide ceramic Al® synthesis was conducted according to the patent [1.2.Preparation of nickel foils with a thickness of 0.1 mm and dimensions of 220 × 120 mm2.4·5H2O), at a current density of 0.02 A/cm2 for 6 h.Galvanic coating of the Ni foil with a layer of copper with a thickness of 100 µm in a solution of copper sulphate pentahydrate (CuSO3.a.Heating in vacuum (10 Pa pressure) up to 1060 °C.b.Carburizing in an atmosphere of acetylene, ethylene, and hydrogen (2:2:1), at a flow rate of 4.0 L/min acetylene and ethylene, and 2.0 L/min hydrogen, consisting of four alternating stages of carbonation and annealing, 5 s of gas dosing/15 min diffusion.c.Heating in an atmosphere of argon and hydrogen to a temperature above the copper melting point (1100 °C) under a pressure of 2 kPa followed by a 5 min soak.d.Cooling the batch to ambient temperature under the same conditions.Graphene synthesis on a Cu-Ni bimetallic substrate in a vacuum generator SuperCarb, Seco/Warwick SA, Świebodzin, Poland, according to the procedure:HSMGe patent . The pro2 in size. The foils were previously washed in an ultrasonic cleaner in isopropanol and then in acetone.The CVD graphene synthesis process on copper solid-state foils was conducted by creating optimal conditions for carburizing in the SuperCarb vacuum generator. An acetylene, ethylene, and hydrogen (2:2:1) gas mixture was used with gas flow rates of 4.0 L/min for acetylene and ethylene and 2.0 L/min for hydrogen. Graphene was synthesized on copper foils 0.1 mm thick and 50 × 50 mm1.Heating in vacuum (10 Pa) to a temperature of 1000 °C,2.Carburizing in an acetylene–ethylene–hydrogen atmosphere for 1 min.3.Heating in a vacuum at 1000 °C for 5 min.4.Carburizing in an acetylene–ethylene–hydrogen atmosphere for 0.5 min.5.Cooling in vacuum to ambient temperature.The process of producing CVD graphene was as follows:2 of graphene. The polymer was dried at a temperature of c.a. 40 °C for 30 min. The double support layer consisted of an additional top layer of poly(dimethylsiloxane), produced by applying about 20 mg of liquid PDMS per cm2 of graphene. The polymer was prepared from a silicone elastomer base and hardener SYLGARD® 184 in a ratio of 10:1. The drying time of the PDMS layer at 60 °C was 60 min. After that, the growth substrate with graphene and polymer was placed in the grip in a 0.5 M NaOH solution so that only its edge was immersed in the electrolyte. The process of graphene delamination was started by applying a constant voltage in the range of 2.5–5 V, causing hydrogen generation between the graphene and growth substrate (cathode). The progress of graphene separation was carried out at a constant speed of 0.01–0.05 mm/s set in the program so that the graphene separation line was at the level of the liquid level. The graphene was then washed twice in distilled water and air dried. Graphene quality was tested by measuring its resistance using the UNI-T UT70B multimeter, UNI-TREND Technology, China, with needle probes.Graphene was separated from the growth substrate using the electrochemical delamination method, which was preceded by drop/blade coating of graphene by a one or two-layer carrier polymer. A 0.05 M solution of polymethyl methacrylate  dissolved in 1 mL of chlorobenzene Chempur Piekary Śląskie, Poland) was used in the amount of 40 μL of solution per 1 cm2O3) was quartz (SiO2). Transfer on quartz is less problematic thus methods presented in the literature were adapted for the needs of graphene transfer to a porous aluminium oxide substrate. However, porous Al2O3 is such a different material that the adoption of the 1:1 transfer methodology resulted in poor quality graphene, the main reason being insufficient adhesion of graphene to porous ceramics. The standard wet transfer procedure with the elimination of the polymer in boiling acetone vapour, which works well in the case of quartz, did not work in this case. Attempts were made to increase the adhesion of graphene to ceramics by applying 2 mL of a 75% acetone/water solution on the sample before placing in it the bath [The reference point in the development of the graphene transfer methodology to alumina ceramics . The system consists of two metal discharge electrodes covered with a dielectric, the so-called dielectric barriers, separated from each other by a 2-millimetre discharge gap. After the system is powered by a high-voltage generator with a frequency of approx. 80 kHz, plasma is generated in the space between the electrodes.In order to improve the adhesion of graphene to ceramics, an attempt was made to change the properties of its surface by creating a barrier discharge powered with a voltage of 15 kV and a frequency of 80 kHz. The time of exposure of the samples to the plasma thus formed was 1 min.2O3 surface with oxygen plasma. For this purpose, the RF PACVD  method was used. The main components of the RF PACVD plasma surface modification equipment are: a working chamber with an HF (high-frequency) electrode, a diffusion pump, a gas dosing system, a pressure measurement system, a radio frequency (13.56 MHz) generator, and a pyrometer for temperature control. The process parameters are shown in In order to increase the adhesion between the transferred graphene and the target ceramic substrate, an attempt was also made to modify the AlChemical etching of the ceramics with an aqueous solution of hydrofluoric acid  was also used to increase the adhesion of graphene to the surface. Solutions of two concentrations were used: 5 and 9.5% HF; pickling time 15 min.2O3 samples were performed.The assessment of the change in wettability of the ceramic surface with distilled water after the modifications was made using a Krüss DSA10 goniometer, Hamburg, Germany. The contact angles were determined based on photos of liquid drops with a volume of 0.8 μL placed on the surface of the samples, taken with a camera set in the axis perpendicular to the surface of the sample. Investigations on non-porous AlTo measure the change in ceramic surface roughness after etching with hydrofluoric acid (HF), a contact profilometer HOMMEL TESTER T1000was used, supported by the Turbo Datawin NT 1.34 software, both from JENOPTIK Industrial metrology, Villingen-Schwenningen, Germany, enabling the determination of surface roughness and waviness parameters based on the obtained etching profiles. Based on the Ra measurement results, the effect of HF acid concentration on the change in the surface topography of the tested samples was determined.−1. Spectra were registered with a resolution of 4 cm−1 with the use of a DTGS detector. The measurements were made in a reflection mode, with the use of Sequelle DRIFT mode, working at a reflection angle equal to 20 degrees. In the case of each material, data from 128 scans were collected in order to construct a single spectrum.The chemical structure was determined by using Fourier-transform infrared spectroscopy (FTIR) using a Thermo Scientific iS50 Spectrometer, Waltham, MA, USA, with a spectral range from 4000 to 400 cmIn order to detect large defects on the surface, the transferred graphene layers were observed at low magnification using a Nikon AZ, Tokyo, Japan stereomicroscope with NIS-Elements software for automatic image registration and processing.2 was analysed each time. In this way, the share of defects in the total area of the analysed sample was calculated.A Nikon Eclipse MA200, Tokyo, Japan light microscope, equipped with NIS-Elements image analysis software, was used to accurately assess the defects in the surface of the transferred graphene in micro-areas. This type of microscope was used because, due to the strong representation of the topography of the ceramic surface, the analysis of the continuity of the graphene layer using a scanning electron microscope, both in the secondary electron (SE) and AEE mode , turned out to be ambiguous. ImageJ software, LOCI, Madison, WI, USA, was used to perform statistical evaluation of graphene defects after transfer onto a porous ceramic substrate. An area of 1 cm−1. Two-dimensional Raman maps were collected from 50 × 50 μm areas with 5 μm spatial resolution. All the collected Raman spectra and maps were preprocessed using WiRE 5.5 software .The Raman spectra and maps were carried out with an InVia Raman microscope  system. The graphene surfaces were exanimated using a 532 nm laser with a 50× objective lens . The laser power at the sample was restricted to 4 mW. For Raman maps, map image acquisition mapping was applied in the wavenumber range of 900 to 3200 cm2O3 ceramics before and after chemical modification was carried out. The analysis of the data presented in 2O3 with a 5% HF acid solution resulted in the levelling of its surface unevenness, manifested by a decrease in the value of the Ra parameter by 0.15 µm compared to its initial state. On the other hand, the almost 2-fold increase in the acid concentration caused a drastic increase in the surface roughness of the Al2O3 ceramics by more than 1µm compared to its state before the modification.To increase the contact area between the surface layer of ceramics and graphene as a result of etching the ceramic surface with 5% and 9.5% hydrofluoric acid (HF) solutions, an analysis of the Ra value of the surface of Al2O3) has weak hydrophilic properties, consistent with the literature data [Investigations of the ceramics’ wettability with the use of distilled water showed that corundum oxide  and the hydroxyl donor ion, which is the so-called Lewis principle. On the other hand, hydroxyl groups behave as Bronsted acid sites. However, high-energy plasma surface cleaning can cause two adjacent OH ions on the alumina surface to form an oxygen bridge, which, however, is the Lewis acid active site, as shown in the diagram below . Such a −1 disappeared almost completely.Moreover, surface modification in plasma also cleans/etches the surface of organic impurities. The band in the range of 3000–2800 cm−1, there was also a slight increase in the intensity of the peaks at the wave numbers 600 and 657 cm−1, which originate from the Al-O bonds characteristic of the alumina structure, which means that the surfaces may have undergone additional oxidation  which is presented in In the range of 1030–400 cm2O3 ceramics. The picture  ceramic substrates using the wet transfer method supported by a PDMS/PMMA layer. Then, a qualitative assessment of graphene on an aluminium oxide substrate was carried out. Images obtained with a stereomicroscope, at 40× magnification, showed the surface of both types of graphene after etching the ceramics with DBD barrier discharge plasma  was carried out under a light microscope. The analysis of the images showed that cleaning the ceramic substrate with oxygen plasma , G (at about 1580 cm−1), and 2D (about 2690 cm−1) peaks. The presented maps show the change in the ratio of D to G peaks on the studied surfaces, which, according to the literature [2O3 ceramics. On the studied surfaces, the lowest, highest, and intermediate degree of defect sites with the lowest (marked “1”), highest (marked “2”), and intermediate (marked “3”) ID/IG ratio values are indicated. The least damaged layer of HSMG® graphene was obtained after transfer to ceramics modified by oxygen plasma (RF PACVD). The most defective HSMG® graphene was on ceramics etched with 5% HF. The same was true for CVD graphene. The least damaged graphene was found on ceramics modified with oxygen plasma and a similar result was obtained for the barrier plasma. The degree of damage to the graphene layer on the substrate etched with 5% HF was the highest.graphene ,33,48,49terature ,49,50,51D/IG ratio was also determined for all points from the tested surfaces. When studying the transferred HSMG® graphene, the average ID/IG ratio was shown to be the lowest for the initial modification of substrates with RF PACVD oxygen plasma (0.63 ± 0.18). When DBD plasma was used, it increased to a value of 0.72 ± 0.17, and in the case of ceramic surface treatment with acids, it was already 0.77 ± 0.28. On the other hand, in the study of CVD graphene, the lowest ID/IG values were obtained for modification with DBD plasma (0.17 ± 0.09), while for samples treated with RF PACVD oxygen plasma or treated with acids, the values were higher and were 0.21 ± 0.08 and 0.21 ± 0.19, respectively.On the basis of the analysis carried out for each sample, the average value of the I2O3 ceramics, it is possible to transfer monolayer HSMG® and CVD graphene to this substrate. The surface modification strongly affects the change of the free energy of the ceramic surface. This surface, especially after plasma modification, became strongly hydrophilic. This had a direct impact on the possibility of transferring graphene sheets. The quality of the transferred graphene, in turn, is affected by the modification method used. The conducted tests showed that the best quality HSMG® graphene layers were the ones transferred to a substrate modified with low-temperature oxygen plasma (RF PACVD). Additionally, for CVD graphene, the best option to modify the surface of ceramics was barrier plasma (DBD). As indicated by the FTIR research, this results from the chemical cleaning of the surface, consisting of the removal of organic impurities, and most of the hydroxyl groups are related to the chemical activation of this surface. FTIR studies of ceramics modified with barrier plasma did not confirm such surface changes as was the case with oxygen plasma. This may be related to the shorter duration of the modification effect in the case of DBD, which did not allow the study of this effect on FTIR. Qualitatively, both HSMG® and CVD graphene were characterized by a low degree of defects after modification with oxygen or barrier plasma. Large damage to the graphene layers, both on a macroscopic and microscopic scale, was observed for samples subjected to chemical modification. This is probably due to the different nature of these changes, which in this case are related to the development of the ceramic surface. A slight effect of the graphene production method on its quality after transfer to ceramics was also found. Research indicates that graphene produced by CVD in this case was characterized by a slightly lower defect degree after transfer to Al2O3. It should be emphasised that the degree of defects in graphene layers is not only related to transfer. Damage to the graphene layers arises first during its synthesis, and again during the transfer. Based on both microscopic and Raman spectroscopy studies (the probed D band), the source of origin cannot be unequivocally indicated.The presented work proves that by modifying the surface properties of Al2O3 ceramics. The preparation of such novel composites could find practical applications as stable membrane materials for the filtration of liquids and gases at extreme temperatures and pH or under high pressure in many industries, such as the biotechnology, pharmaceutical, food, chemical, and petrochemical industries. However, for their practical use in filtration, further research is needed to assess their effectiveness. This work focused primarily on the development of an effective method of transferring large-area graphene to a porous ceramic substrate with Al2O3 and assessing its quality after transfer on a macro- and microscopic scale. Further research should include the assessment of graphene defects on the ceramic substrate at the nanoscale and the determination of the filtration characteristics of these membranes.In conclusion, the obtained research results allow for obtaining composite membranes based on graphene and Al


Given the potential large ethical and societal implications of human germline gene editing (HGGE) the urgent need for public and stakeholder engagement (PSE) has been repeatedly expressed. In this short communication, we aim to provide directions for broad and inclusive PSE by emphasizing the importance of futures literacy, which is a skill to imagine diverse and multiple futures and to use these as lenses to look at the present anew. By first addressing “what if” questions in PSE, different futures come into focus and limitations that arise when starting with the “whether” or “how” questions about HGGE can be avoided. Futures literacy can also aid in the goal of societal alignment, as “what if” questions can be answered in many different ways, thereby opening up the conversation to explore a multitude of values and needs of various publics. Broad and inclusive PSE on HGGE starts with asking the right questions. Decisions about whether or not to pursue human germline gene editing (HGGE; Table PSE has the potential to enable policy making that is informed by the values and needs of society at large (Scheufele et al. Inclusivity is described as one of the ideals for effective PSE in developing HGGE policies (Iltis et al. Bucchi and Trench  defined ‘Globally’, as Françoise Baylis writes, ‘the political consensus on heritable human genome editing—such as it is—inclines toward an outright ban, and if not a ban, at least a moratorium’ Baylis . AlthougThe “what if” questions require time for the public to reflect, slow down, use imagination and think beyond their current lived experiences. They allow the public to think about the ways in which HGGE could improve their lives and the lives of their children, as well as which futures would be undesirable and inconsistent with their values. This speculative “if yes, then how” question should not be confused with the question of “how” HGGE should be implemented. Therefore, it is important to be transparent about this distinction at the outset of PSE. The questions “whether” and “how” provide valuable insight in how practice and policy around HGGE should be organized, which will become relevant once the scenarios of what our society may become are deliberated on. Until then, there is a widely shared responsibility to enable this deliberation and opinion formation (Houtman et al. Broad and inclusive social conversation around HGGE starts with asking the right questions. By focusing on questions of “what if” before “whether” and “how” in PSE we can open up the conversation by creating various future narratives. Consequently, we reduce directivity and framing as initiators of PSE, and increase inclusiveness as this approach does not exclude in advance those who hold fundamental objections against HGGE or those who have yet to make up their mind for various reasons. Actual participation in PSE by the aforementioned groups is something that should be evaluated and monitored continuously. Asking “what if” will surface the values and needs that are at stake, which can then be taken into consideration when the questions of “whether” and possibly “how” are deliberated on.


BackgroundEpistaxis is a very common symptom. The occurrence of epistaxis may be affected by dry environments, but there are some differences among previous reports and this view is controversial.ObjectiveWe investigated the relationship between the number of epistaxes and daily average relative humidity.MethodsData on patients with epistaxis between March 2011 and February 2021 were collected from two hospitals. The daily average relative humidity was examined, and the change in the number of patients with epistaxis due to humidity was investigated using a generalized linear mixed model.ResultsA total of 4184 cases of epistaxis were identified. The number of epistaxis cases per day was significantly associated with the daily average relative humidity (p < 0.001). One percent increment in average relative humidity decreases the number of epistaxis cases per day by 1.1%.ConclusionA negative correlation was found to exist between daily average relative humidity and occurrences of epistaxis. Epistaxis is one of the most common complaints in otolaryngology. Although the detailed pathological causes of epistaxis are unknown, it is commonly believed that epistaxis occurs during the colder, drier months . SeveralPatientsThe subjects of this study were all patients at Tsukuba University Hospital Mito Medical Center, Mito Kyodo General Hospital, Mito, Japan  and Tsukuba Gakuen Hospital, Tsukuba, Japan , two important acute care hospitals in Ibaraki Prefecture, Japan. The patients were diagnosed with epistaxis from March 2011 to February 2021. The data items collected from each hospital included disease name, age, sex, and date of diagnosis. For statistical analysis, the date of diagnosis was considered as the date of onset of epistaxis.The study was carried out in accordance with the tenet of the Declaration of Helsinki and the work was compliant with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. The institutional review boards of the respective participating institutions approved this study . By opting out, the institutional review board exempted this retrospective study from the requirement of obtaining informed consent from the individual participants.Meteorological datahttps://www.jma.go.jp/jma/index.html). The average relative humidity data was adopted for each hospital area. The number of days with the same humidity (%) over a 10-year period was counted. In addition, the number of patients with epistaxis who visited on days with the same humidity (%) was also counted.The average relative humidity values analyzed in this study were obtained from the Japan Meteorological Agency website  for categorical variables, and as median (range) for continuous variables. A generalized linear mixed model with Poisson distribution and log link function was used to explore the association between the incidence of epistaxis and average relative humidity, after adjusting for the number of days with the same humidity. In the model, the response variable was the number of patients per day and the fixed effect was the average relative humidity. The hospital was used as a random effect. All statistical analyses were performed with SAS version 9.4 .Patient characteristicsOverall, 4184 cases of epistaxis at the participating centers were enrolled in this study. The median age of the patients was 63 years (range: 0 to 97 years). Of the patients, 2520 (60.2%) were male and 1664 (39.8%) were female. The sex ratio and median age were comparable between the two facilities . One percent increment in average relative humidity decreased the number of epistaxis cases per day by 1.1% Figure .In this study, we observed the negative correlation between daily average relative humidity and the number of patients with epistaxis by examining a large amount of data from two hospitals over 10 years. One percent increment in average relative humidity decreased the number of epistaxis cases per day by 1.1%. That is, a 10% increase in average relative humidity resulted in a 10.5% decrease in the number of patients with epistaxis, and a 20% increase resulted in a 19.8% decrease.Several studies have investigated the relationship between humidity and epistaxis -7. The mEpistaxis has a bimodal age distribution, being more common in children under the age of 10 and adults in their 70s, and occurs more frequently in males . One of It is difficult to identify a pathophysiological explanation as to why a decrease in humidity increases the risk of epistaxis. The nasal gland system, stimulated by dry air, protects the lower airways by humidifying the air by producing a variety of secretions . The venIn this study, due to the large sample size, we could not review individual charts; therefore, cases of varying severity were included. Information on treatment was not available in the database used for the study. The date of diagnosis of epistaxis was investigated; however, the date of occurrence and the date of diagnosis may not necessarily be the same. Moreover, although our study included the most important acute care hospitals in the region, the extent to which the results of this study can be generalized to other countries with different environments is limited. Despite these limitations, this study provides strong support for the hypothesis that dryness is a risk factor for epistaxis. Our result may be useful for patients who are prone to epistaxis and additional research is needed.In this study of a large population in a specific area belonging to the humid subtropical climate, there was a strong negative correlation between daily average relative humidity and the number of patients diagnosed with epistaxis in selected hospitals. This result may be related to increased intranasal resistance and decreased mucociliary movement due to dry air. This result supports the hypothesis that dryness is a risk for epistaxis.


Emergency departments are key settings for suicide prevention. Most people are deemed to be at no or low risk in final contacts before death.To micro-analyse how clinicians ask about suicidal ideation and/or self-harm in emergency department psychosocial assessments and how patients respond.Forty-six psychosocial assessments between mental health clinicians and people with suicidal ideation and/or self-harm were video-recorded. Verbal and non-verbal features of 55 question–answer sequences about self-harm thoughts and/or actions were micro-analysed using conversation analysis. Fisher's exact test was used to test the hypothesis that question type was associated with patient disclosure.N = 46/55) were closed yes/no questions about self-harm thoughts and/or feelings, plans to self-harm, potential for future self-harm, predicting risk of future self-harm and being okay or keeping safe. Patients disclosed minimal information in response to closed questions, whereas open questions elicited ambivalent and information rich responses. (b) All closed questions were leading, with 54% inviting no and 46% inviting yes. When patients were asked no-inviting questions, the disclosure rate was 8%, compared to 65% when asked yes-inviting questions (P < 0.05 Fisher's exact test). (c) Patients struggled to respond when asked to predict future self-harm or guarantee safety. (d) Half of closed questions had a narrow timeframe  or were tied to possible discharge.(a) Eighty-four per cent of initial questions (Across assessments, there is a bias towards not uncovering thoughts and plans of self-harm through the cumulative effect of leading questions that invite a no response, their narrow timeframe and tying questions to possible discharge. Open questions, yes-inviting questions and asking how people feel about the future facilitate disclosure. Meta-analysis indicates that only 28.7% of people disclose suicidal intent to healthcare professionals before suicide.2 Many patients are reported to ‘deny suicidal ideation’ in the days or weeks before a suicide attempt.4 Importantly, UK professionals judge immediate risk of suicide to be low or not present in 85% of assessments in the final appointment before death.5 Self-harm, self-injury or self-poisoning irrespective of intent6 is the strongest risk factor for suicide.7 Most studies have focused on professional reports or patients’ medical records rather than direct research on communication about self-harm.Suicide prevention is a global public health priority, with over 700 000 deaths by suicide annually.8 Self-harm remains highly stigmatised, and patients report shame, guilt, fear about loss of autonomy9 and onward consequences such as admission or removal of children from their care.10Communicating about self-harm is challenging. Sociological studies have shown a strong preference for ‘saving face’ and not disclosing face-threatening and stigmatised information in social interactions.11 e.g. ‘Are you feeling suicidal?’ is framed positively, inviting agreement to ‘feeling suicidal’.12 Conversely, ‘Not feeling suicidal?’ is negatively framed, inviting agreement to ‘not feeling suicidal’. Word choice further reinforces the direction of the question. Words such as ‘any’, ‘ever’, ‘at all’ are negative polarity items  that invite a ‘no self-harm’ response and affect patients’ likelihood of responding ‘no’.13Recent studies have highlighted that clinicians’ questions about self-harm can have a considerable impact on disclosure of thoughts and plans. Yes/no questions are prevalent in medical interaction. Although medical communication training encourages professionals to ask non-leading questions, there is no such thing as a non-leading yes/no question. Yes/no questions communicate an expectation in favour of either ‘yes’ or ‘no’ responses through wording and grammatical structure,14 In secondary out-patient mental healthcare, more than 75% of questions invited patients to confirm the absence of self-harm thoughts and/or actions.15 Patients were significantly more likely to say no in response to no-inviting questions (compared with yes-inviting questions), even though some disclosed suicidal thoughts in self-report measures.In primary care, most questions invite ‘no self-harm’ answers, with patients showing difficulty reporting self-harm thoughts and/or actions when clinicians ask these leading questions .6 around 220 000 episodes of self-harm by 150 000 people are managed by emergency departments in England annually.16 Most emergency departments have a psychiatric liaison team staffed by specialist mental health clinicians. They conduct psychosocial assessments after self-harm and make onward referrals.6 Emergency departments are high-pressure environments with 4 h targets for patients to be seen, treated and admitted or discharged. Although the number of people seeking help for mental health problems has risen year on year, numbers of hospital beds have fallen,17 increasing the pressure to discharge patients. To date, no studies have observed psychosocial assessments for self-harm in emergency departments. This study addressed the following questions.How do clinicians ask patients about suicidal ideation and/or self-harm in emergency department psychosocial assessments?How do patients respond verbally and non-verbally?A key setting for suicide prevention is the emergency department:This was a cross-sectional non-participant observational study of clinical practice, involving video-recording psychosocial assessments between people presenting to the emergency department for suicidal ideation and/or self-harm and psychiatric liaison clinicians.6, providing the basis for a decision on whether to discharge and a management plan for further support.The study took place in an emergency department in England. As per usual practice, people seeking care for suicidal ideation and/or self-harm met with emergency department triage staff to assess clinical urgency and undergo any physical interventions before referral to the emergency department liaison psychiatry service for a psychosocial assessment, including risk of harm to self18All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures were approved by London Central Research Ethics Committee (17/LO/1234). Study design, materials and recruitment processes were developed in collaboration with a lived experience group including one carer, one mental health nurse and six people who had presented to the emergency department for suicidal ideation and/or self-harm. Written informed consent was obtained from all participants.N = 11), a student nurse (N = 1), doctors (N = 2), junior doctors (N = 7), social workers (N = 2), occupational therapists (N = 2), healthcare assistants (N = 2), consultant psychiatrists (N = 2), psychiatry trainees (N = 2) and unspecified (N = 2). Their mean age was 38.3 years , and they were mostly female (N = 20/33) and White British (N = 28/33).All 43 psychiatric liaison clinicians in the psychiatric liaison team who conducted psychosocial assessments were invited to participate, and 33 consented (76.7%). Clinician participants comprised mental health nurses , self-harm by overdose (N = 23/46) or self-harm by ligature or attempted drowning (N = 3/46). Patient mean age was 35.5 years . They were mostly female (N = 31/45) and White British (N = 43/45). Caregivers, typically parents, were involved in eight assessments.Recruitment took place from September 2018 to April 2019. A total of 260 referrals were screened, 82 individuals were approached regarding participation and 48 consented. Three patients were excluded after consenting, as they did not present with suicidal ideation or self-harm. One patient re-presented, resulting in a total of 46 assessments. Referrals were for suicidal ideation  as the focus was on initial disclosure of thoughts, actions or plans. Questions about acting on thoughts or repeating self-harm were typically asked at the end of the assessment, alongside questions about next steps and discharge.19 to micro-analyse clinicians’ questions and patients’ responses. Standardised methods were used to transcribe and analyse the content of speech and characteristics such as pauses, overlap, stress, intonation, pace and non-verbal communication.20 This paper contains simplified transcripts with names and locations changed. Numbers in brackets denote silence in seconds. We coded the following.(i) open or closed20(ii) inviting a yes or no responseClinicians’ questions as:no self-harm responseself-harm response21‘non-answer’ that did not provide an answer to the question, e.g. ‘I don't know’23resisting the question or an assumption in the question, e.g. ‘I'd like to say no but that's one of those things that – I – I don't think I could guarantee it’.Patients’ responses as:How the question affected the patient's response. As previous work has shown that questions bias patients’ responses, we used Fisher's exact test to test the hypothesis that no-inviting questions were more likely to elicit ‘no self-harm’ responses.We used a qualitative method, conversation analysis,24 Disengagement was classified as ≥3 markers of disengagement.In the videos, patients did not always show conviction or certainty in responses that claimed ‘no suicidal ideation’ or ‘no self-harm’. Hence, we identified (a) statements indicating lack of knowledge or certainty (e.g. ‘I don't know’), and (b) signs of patient disengagement, which could indicate difficulty or reluctance to disclose more sensitive information. This included flat prosody, quiet or slow speech relative to the patient's typical speech, minimal lexical responses (e.g. ‘No. [2 s]’), averting gaze, flat or unchanging facial expression, and physically turning away from the clinician.N = 19), plans to self-harm (N = 13), the potential for future self-harm (N = 13), predicting risk of future self-harm (N = 5) and being okay and/or keeping safe (N = 5).There were 55 initial questions about current thoughts or feelings of self-harm (N = 46/55). However, in 16% cases (N = 9/55), initial questions were open questions, starting with ‘what/when/how’, inviting the patient to provide a longer response . For example:Video extract 1Clinician:  So, do you have any thoughts of ending yourlife as we speak now?Patient:    (2 s)No. (1 s)Video extract 2Clinician:  And are you feeling suicidal?Patient:   (2 s)Mhm. Yeah. (4 s)Video extract 3Clinician:  You'd take it [overdose] again if you could?Patient:   nods (1 s)Closed questions ask patients to choose yes or no, and are designed to constrain responses. Patients typically provided minimal answers followed by silence and did not elaborate on their experiences (Video extract 4Clinician: What's your thoughts about wanting to die?Patient:  (1 s)They're, I still kind of want to die. Butthen Jack thinks that I can't die ‘cause I gotmy boyfriend and there are people who care aboutme. (1 s) It's hard. I'm sort of in the middle.Video extract 5Clinician: How do you feel about ending your life as wespeak now?Patient:  It's still on the forefront of my mind. This seemslike a sticking plaster coming up here. And (1 s)I'm afraid to go home and be on my own again.Because I think there'll just be another day whereI'm not strong enough to override it and call forhelp and they'll – they'll just find me.Video extract 6Clinician: What plans do you have now?Patient:   Like I'm not prepared for the pain that I knowthat ligaturing is gonna cause. Like that'sgonna be painful, I know it is.By contrast, open questions elicited longer information-rich responses, with patients frequently describing conflicting or ambiguous thoughts.12 Of 46 closed questions, more than half  invited a no-self-harm response, with fewer than half (46%) inviting a yes-self-harm response. No-inviting questions used the negative polarity markers any/ever/at all  compared with the 65% disclosure rate in response to yes-inviting questions   .Table 325 For example, in extract 7, there are long delays , the patient breaks eye contact (line 3) and eventually confirms (‘I do’) but downplays the extent (‘a few’) and does not describe his ideas or plans in any detail.Video extract 7Clinician: Can I ask (1 s) Do you have any        plans todo anything to harm yourself?(2 s) patient looks downClinician: Or to take your life?(4 s)Patient:(no eye contact) I do have a few ideas.(1 s)There is further evidence on how patients respond to no-inviting questions demonstrating that such questions are highly problematic for disclosing suicidal ideation and/or self-harm: patients often delayed their response, qualified or downplayed their response, looked away and provided minimal information. These features mark difficulty responding.24 Patients gave a disengaged response 35% of the time (N = 7/20) when giving a no-problem answer to a no-inviting question versus 11% of the time (N = 1/9) when giving a no-problem answer to a yes-inviting question.Video extract 8Clinician: You're seeing [your therapist] tomorrowaren't you?Patient:   Yeah.(3 s)Clinician: You- Are you gonna be okay overnight?Patient:   patient looks down(1 s)nods(2 s)Second, when patients did not disclose suicidal ideation or self-harm, they more frequently showed signs of disengagement and lack of conviction after no-inviting questions. For example, after being asked if she will be okay overnight, the patient in extract 8 confirms but immediately shows signs of disengagement: breaking off eye contact, looking at the floor and nodding silently.N = 19/55, 35%)  .Table 4N = 12) of these questions inviting the patient to confirm they would not be at risk or could keep themselves safe. Asking for a prediction is different to asking patients whether they currently have plans or thoughts about ending their life (e.g. ‘Do you have any plans to do this again?’).Again, there was a bias with 63% (Video extract 9Clinician: What's the rating that you would give – probability thatyou'll take another overdose?Patient:     (5 s)I don't knowExplicitly claiming lack of knowledge:Video extract 10Clinician: What do you think the chances are of you actually goingthrough with some of those suicidal thoughts that you havePatient:    (4 s)I think (3 s)My – You know – (2 s)couldn't do it to my momI thinkHedging their response using ‘I think’, false starts and cut-off words (‘My-’) and downgrading their certainty:Patients found these questions particularly difficult to answer, as evidenced by substantial delay and resisting the implication that they could predict future self-harm in the following ways.Video extract 11Clinician: You think you'll do it again?Patient:  (2 s)I'd like to say no but that's one of those things that – I –I don't think I could guarantee it.Patients also emphasised the difficulty of assessing the likelihood of future suicidal acts when this was something they did not want, e.g. in extract 11 the clinician asks if the patient thinks he will attempt suicide again.Instead of giving a straightforward yes or no as invited by the question, the patient states ‘I'd like to say no’. He then gives a hedged answer, citing his uncertainty ‘I don't think I could guarantee’ and difficulty predicting whether he will attempt suicide.Video extract 12Clinician: So is there part of you that's scared you will hurtyourself?Patient:     Yeah.By contrast, there are three instances where a clinician asks about the future without asking the patient to predict their future actions. In extract 12, the clinician asks whether the patient is scared she may hurt herself. She immediately confirms, without hesitation, backpedalling or uncertainty, showing no difficulty in making this disclosure.current thoughts/feelings of self-harm, e.g. ‘So are you thinking about ending your life at the moment?’ ‘Do you have any plans of ending your life once you leave here?’nine of 19 questions about plans to self-harm, e.g. ‘Have you got any plans at the moment to take another overdose or harm yourself?’four of 13 questions about potential for future self-harm, e.g. ‘Where are you at over the next twenty-four hours about hurting yourself?’one of 13 questions about the predicting risk of future self-harm, e.g. ‘And so do you think there's any chance of you doing something else to harm yourself today tomorrow?’two of five questions being okay, e.g. ‘Are you gonna be okay overnight?’two of five questions about Some questions were also temporally constrained with a narrow timeframe (18 of 46 closed questions), as follows:plans to self-harm, e.g. ‘If you were to go home, to do something that would result in your death? Or harm to yourself?’two of 13 questions about potential for future self-harm, e.g. ‘On a basic level if you went home now, would you do anything to harm yourself?’two of 13 questions about keeping safe, e.g. ‘If you were to go home do you think that you could keep yourself safe?’two of five questions about In addition, towards the end of the assessment, some questions about future self-harm were explicitly tied to possible discharge (6/46 closed questions):This placement indicates to the patient that their answer is tied to discharge, which is likely to affect how patients respond if they wish to be discharged.Across the psychosocial assessment, there is a bias towards not uncovering thoughts and plans of self-harm through the cumulative effects of leading questions that invite a no response, their narrow timeframe and tying questions to possible discharge. First, 84% of questions were closed yes/no questions inviting minimal yes/no responses. Second, more than half were leading questions inviting a ‘no’ self-harm answer. When asked no-inviting questions, patients disclosed self-harm in only 8% of cases compared with 65% when asked a yes-inviting question. When answering ‘no’ (e.g. no thoughts or plans of suicide) to no-inviting questions, patients often showed signs of disengagement and lack of conviction (35%). Third, approximately one-third of questions asked patients to predict whether they would harm themselves in the future. Patients showed difficulty responding to these questions, resisting the assumption that they could predict future thoughts or actions, which is underpinned by patients’ criticisms of these questions, including ‘guaranteeing safety’ questions. Finally, half of closed questions had a narrow timeframe  or were explicitly tied to possible discharge.26 Nonetheless, these findings may help to shed light on the ‘low-risk paradox’, i.e. that patients are usually judged as low or no risk prior to death by suicide27 and ‘deny suicidal ideation’ even in the days and weeks before death.4 Our findings highlight that communication is structured in a way that makes it difficult for patients to disclose self-harm. Clinicians are unaware that a subtle difference in the wording of questions biases the patient's response with the items ‘any, ever, at all’ making questions no-inviting. The narrow timeframes demonstrate that clinicians are focusing on short time periods, typically up to 24 h. At the same time, they are tying patient responses to discharge, i.e. if the patient states they have no immediate thoughts or plans they are safe to discharge. Given that most patients report wanting to leave the emergency department after long waits to be seen in the main emergency department before being referred and then undergoing a psychosocial assessment, there is a risk of patients saying what they need to say so they can leave, i.e. they are not thinking about or planning self-harm. As a whole, these findings shed light on the National Confidential Inquiry findings that of the 17 people who die by suicide each day in the UK, four out of five are judged to be at low or no risk of suicide.Suicide risk assessment is a multifactorial process, and assessment of self-harm is just one component.15 In suicide risk assessment, no-inviting questions are compounded by patient shame, stigma and the consequences of disclosing self-harm . Concurrent feelings of wanting to live and die and fluctuating feelings about the ability to manage suicidal thoughts can also make these questions difficult to answer. On the other hand, when clinicians asked open questions, patients disclosed more information, particularly around inconsistent or ambiguous feelings of self-harm. Clinician judgement about the likelihood of self-harm could inform how practitioners design their questions. In conversation analysis, this process of tailoring our talk for the person we are speaking to is known as ‘recipient design’. Although this may contribute to how questions are designed, in a previous study we found that practitioners were not more likely to ask a yes-inviting question of patients who self-reported more suicidal thoughts and/or behaviour14.These findings are consistent with studies in out-patient community mental healthcare and general practice, where professionals also mostly asked no-inviting questions.12 In a randomised controlled trial, doctors who asked ‘Do you have some other concerns you would like to discuss?’, inviting a yes, versus ‘Do you have any other concerns you would like to discuss?’, inviting a no, were significantly more likely to elicit and reduce unmet concerns when using yes-inviting questions.13No-inviting questions are not just a feature of suicide risk assessment but a feature of healthcare questioning more widely. Doctors systematically ask questions inviting the ‘best case’ answer (e.g. ‘No pain?’); this is known as the principle of optimisation.28 This includes making recommendations (e.g. discharge) because of limited resources as opposed to their clinical judgement. This can manifest in burnout, clinician turnover, or developing a more detached or even callous approach to care as a defence mechanism against the system's impact on their practice.Emergency department psychiatric liaison clinicians are faced with increasingly limited options for treatment . If a person ends their life, clinicians may be called to a coroner's court if a person deemed at risk does not receive adequate treatment. Anecdotally, clinicians report defensive practice and can experience ‘moral injury’ when they are expected to work in ways that contradict their moral compass.28 along with adequate supervision and training.29Nonetheless, our findings have clear clinical implications. Patients are more likely to disclose self-harm when asked yes inviting  questions. No inviting questions typically use the words ‘any’, ‘ever’ or ‘at all’  or a negative declarative format (e.g. ‘You're not planning on doing it again?’). Open questions e.g. ‘How do you feel about ending your life as we speak now?’, ‘What are your thoughts about wanting to die?’ yield rich responses on the type and strength of thoughts and reasons the patient would not (re)attempt self-harm. Categorising self-harm thoughts and/or plans as either yes or no is problematic, as most people have ambivalent feelings. Guidelines and training should highlight how subtle differences in the wording of questions significantly influence patient disclosure of suicidal ideation and self-harm. For real change, there must also be an understanding of how clinicians protect themselves and cope with the challenges of underresourcing,N = 21/45) and 60% were female. Further research with minority ethnic groups, different socioeconomic groups and other marginalised groups to look at assessments in these groups in more detail would be important to understand whether communication differs and, if so, how it differs. We focused on initial questions as they influence the trajectory of further enquiry. We did not focus on caregiver communication, which was beyond the scope of this analysis. This is the first detailed analysis of video-recorded communication about self-harm in the emergency department. Conversation analysis allowed us to analyse non-verbal communication and to identify how subtle differences in question design affect patient disclosure.Data were collected in one emergency department, which may limit the generalisability of the findings. However, the findings are consistent with communications about self-harm in secondary and primary care. Around three-quarters of clinicians and just under 60% of patients consented: those consenting may not be representative of clinicians and patients. As is typical of many studies, patient participants were mostly White British (In conclusion, there is a bias towards not uncovering self-harm thoughts and plans in the emergency department. Closed no-inviting questions and asking patients to predict the future are common and deter disclosure. Other problematic aspects are narrow timeframes and tying questions to possible discharge. Open questions and yes-inviting questions facilitate disclosure. Asking patients how they feel about the future rather than asking them to predict whether they will harm themselves also facilitates disclosure.


We report the genome sequences of two genetic variants of grapevine rupestris stem pitting-associated virus (GRSPaV) from Idaho, USA. The coding-complete, positive-strand RNA genome of 8,700 nucleotides contains six open reading frames characteristic of foveaviruses. The two Idaho genetic variants belong to GRSPaV phylogroup 1. Foveavirus (family Betaflexiviridae) (Vitis vinifera L. and its hybrids (––Grapevine rupestris stem pitting-associated virus (GRSPaV) has flexuous filamentous particles and belongs to the genus viridae) . GRSPaV viridae) . The vir hybrids –4. The vhybrids –, 5, 6; ihybrids ––9.V. vinifera reference genome using bowtie2 v2.4.4 in local mode  from British Columbia, Canada. The same Riesling sample also yielded three additional contigs of 2,514, 2,893, and 506 nt, which exhibited 98.3%, 98.6%, and 98.8% identity, respectively, to the GRSPaV isolate AMCF clone 3 (GenBank accession number MG938311) from France  contig (GRSPaV-ID1) that spanned six ORFs and, based on the BLASTn program , exhibited 98.5% identity to the GRSPaV isolate 12G412  under BioProject accession number PRJNA931750.The coding-complete genome sequences of GSPaV-ID1 and GSPaV-ID2 are available in GenBank under accession numbers


In vitro culture, ensuring rapid multiplication and production of plant material under aseptic conditions, represents an excellent tool for ex-situ conservation of tree species biodiversity and can be used for the conservation, among others, of endangered and rare crops. Among the Pyrus communis L. cultivars that have been abandoned over the years due to changed cultivation requirements, but which are still used today in breeding programs, there is the ‘Decana d’inverno’. Pear is generally considered a recalcitrant species for in vitro propagation due to weak multiplication rate, hyperhydricity, and susceptibility to phenolic oxidation. Therefore, the use of natural substances like neem oil  represents one of the options to improve the in vitro plant’s tissue culture. In this context, the aim of the present work was to evaluate the effect of adding neem oil (0.1 and 0.5 m L L−1) to the growth substrate in order to optimise the in vitro culture of the ancient pear tree cultivar ‘Decana d’inverno’. The neem oil addition resulted in an increase in the number of shoots produced especially at both concentrations used. On the contrary, an increase in length of proliferated shoots was observed only with the addition of 0.1 mL L−1. The neem oil addition did not affect the explants viability, fresh and dry weights. Therefore, the present study demonstrated for the first time the possibility of using neem oil to optimise the in vitro culture of an ancient pear tree cultivar. Pyrus is characterized by a high genetic variability with several species and thousands of cultivars that can be divided into two major groups, the Occidental (European) and the Oriental (Asian) pears [The genus n) pears . Regardin) pears .Valsa pyri are increasing [The world production of pears in 2021 was 25.6 Mt across a cultivated area of 1.4 million hectares (ha) . The lar® was obtained in 1994 from the INRA by crossing ‘Decana del Comizio’ × ‘Decana d’inverno’, while ‘Cepuna’ was obtained by crossing ‘Conference’ and ‘Decana d’inverno’ [in vitro culture [In vitro cell and tissue culture techniques do indeed ensure the rapid multiplication and production of plant material under aseptic conditions and can be used for the conservation among the others of endangered and rare crops [In vitro culture has been reported for several cultivars/rootstock of pear [in vitro propagation (micropropagation) [in vitro plant’s tissue culture [in vitro growing media there is neem oil. Previous researches [in vitro culture of other plants species. Neem oil is obtained from Azadirachta indica A. Juss seeds and thanks to its composition its widely used in agriculture and for human health [in vitro culture of an ancient pear tree cultivar (‘Decana d’inverno’).In addition, there is a lack of varietal innovation in all the growing countries. Indeed, in Europe, the mostly diffused pear cultivar is Conference, followed by Abbé Fétel, Williams, and Rocha, while in Italy, one of the world’s largest producers of pears, a large part of production is based on two main cultivars, Abbé Fétel and Williams . Among tinverno’ . In this culture ,8. In vi of pear ,14,15,16agation) . In this culture . Among tsearches  demonstrn health . Neem oin health . In partn health ,23. In t−1) (−1 (ON 0.1) and 0.5 mL L−1 (ON 0.5) of neem oil were added an increase of about 34% and 30% respect to the control (0 mL L−1 of neem oil) in the number of shoots was observed. On the contrary, a positive effect on length of shoots was detected only when neem oil was applied at 0.1 mL L−1. In the ON 0.1 substrate an increase in shoot length of about 30% was observed while at 0.5 mL L−1 neem oil did not induce an increase in shoot length.Neem oil does not affect proliferated explant’s viability. In all treatments, the shoot viability of cv ‘Decana d’inverno’ was equal to 100%. Adding neem oil to the culture medium allowed to obtain a better vegetative activity evaluated as number of shoots produced . The inc−1) . Indeed,−1 (ON 0.1) increased the callus fresh weight. On the contrary, the above-mentioned effect was not detected in the ON 0.5 culture media. The addition of neem oil did not affect green fresh weight and total dry weight of the proliferated explants ’ rootstock in vitro proliferation was obtained thorough a proper combination of plant growth regulators in the MS substrate: a combination of 2 mg L−1 6-Benzylaminopurine (BAP) and 1 mg L−1 Kinetin [−1 BAP [Pear tree is generally considered recalcitrant for micropropagation since during ion rate ,17. Howeexplants  and was explants . To the explants . Thanks explants . Improveexplants  while inexplants . On the  Kinetin . Evaluat [−1 BAP .in vitro Biotechnology of the Tree Science Research Unit of the Department of Agricultural Food and Environmental Sciences of the University of Perugia, where the experiment was conducted. The culture of ‘Decana d’inverno’ was stabilized aseptically, from meristematic apices taken from portions of vegetative resting branches, cut into pieces with a length of 3–4 cm (2–3 nodes), washed with running tap water, and immersed for 10 s in a solution of 70% ethyl alcohol with the addition of a few drops of surfactant (Tween 20) to remove surface impurities. The plant material was then immersed in a 2.5% sodium hypochlorite solution for 20 min and immediately transported under a laminar flow cabinet to operate under completely aseptic conditions. The explants were then washed twice in sterile distilled water to remove residues of the sterilizing agent. The meristematic apices of 0.3–0.5 mm length and approximately 0.1 mm in diameter were then removed under aseptic conditions using a stereomicroscope.The plant material used in the experiment was derived from shoots of the proliferating cultivar Decana d’inverno present at the Laboratory of Micropropagation and −1), hydrolyzed casein (100 mg L−1), agar provided by Duchefa (0.7 g L−1), pH of 5.5. Before exposing the apices to light, the tubes were covered with aluminium foil and kept in the dark in the growth chamber for 7 days to avoid photo-oxidation. The healthy (uncontaminated) apices were kept in the growth chamber at 22 ± 2 °C and gradually accustomed to light intensities of 40 µE m−2 s−1 and a 16-h photoperiod of light. The stabilization phase was completed after approximately 30–35 days. The obtained proliferated shoots were then transferred in glass vessels (500 mL capacity), each containing ten explants and 100 mL of MS substrate enriched with vitamin mixture of Jacquiot instead of MS vitamin mixture [−1), naphthalenacetic acid (NAA) (0.1 mg L−1) and BAP (2 mg L−1), sucrose (30 g L−1) and agar (7 g L−1), pH 5.5.Finally, the apices were transferred into glass tubes each containing 5 mL of Murashige, and Skoog (MS) substrate , supplem mixture , gibbere−1 referred as ‘control’, 0.1 mL L−1 referred as ‘ON 0.1′, and 0.5 mL L−1 referred as ‘ON 0.5′) were added to the substrate before autoclaving at 115 °C for 20 min. In particular, a neem oil soluble in water provided by “Neem Italia company” composed by neem seed oil (from organic agriculture) and Polysorbate 85 was used. The placement of the explant inside the vessels occurred in aseptic conditions under a horizontal laminar flow cabinet. The explants were placed for 30 days in a growth chamber, characterized by a constant temperature of 22 ± 2 °C and a 16-h photoperiod of light with an intensity of 40 µE m−2 s−1.The initial explants for the experiment were represented by 10 mm about long uninodal explants deprived of the apical bud to maintain a high degree of uniformity. Glass vessels (500 mL capacity) were used in the experiment, each containing ten explants with two leaves and 100 mL of the above-mentioned substrate. Following previous experiences conducted on other species, three different neem oil concentrations  equal to six was foreseen: this allowed, at the end of the proliferation, to carry out the destructive measurements on the shoots of three pots and to assure the suitable number of explants for the next subculture.viability (%): incidence of green and viable explants;shoots (n): average number of shoots developed by each initial explant (multiplication rate);shoot length (mm): average length of developed shoots;callus (%): incidence of explants that produced basal callus;green fresh weight (mg): average fresh weight of developed vegetative organs ;callus fresh weight (mg): average fresh weight of callus masses that may have developed at the base of explants;total dry weight (mg): average dry weight of the vegetative organs and callus, obtained by keeping the plant material in an oven for three days at 105 °C.To evaluate the effect of n.o. treatments, after 30 days, the following parameters for each of the three subsequent subcultures were monitored:p = 0.05).The trial was organized according to a completely randomized design, with three treatments and 30 proliferated explants for each treatment and for each of the three subsequent subcultures. In particular, for each treatment and subculture, a number of vessels (replicates) equal to six was foreseen: at the end of the proliferation, this allowed to carry out the destructive measurements on the shoots of three pots (30 proliferated explants for each treatment) and to assure the suitable number of explants for the next subculture. Collected data were subjected to one-way variance analysis (ANOVA), and significant differences were assayed by Tukey HSD test (−1, allows to optimize the in vitro culture of ‘Decana d’inverno’ an ancient pear tree cultivar. The increase in the number of shoots produced and their length allow to increase the efficiency of the micropropagation process and to overcome some issues of the in vitro culture of a species considered recalcitrant.The obtained results suggest that the addition of neem oil, a natural substance, at 0.1 mL L


Sarcoidosis is a non-necrotizing granulomatous inflammatory multisystemic disorder of unknown etiology. In children, as in adults, it can involve a few or all organ systems to a varying extent and degree, entailing multisystemic manifestations. Kidney involvement in pediatric-onset adult-type sarcoidosis is rare, with a wide range of renal manifestations, most of them related to calcium metabolism. Children with renal sarcoidosis tend to be more symptomatic than adults, although male patients have a higher prevalence. We present the case of a 10-year-old boy who presented with advanced renal failure with nephrocalcinosis and important hepatosplenomegaly. The diagnosis was established by histopathological examination, with consequent cortisone therapy and hemodialysis. This review emphasizes that sarcoidosis should be considered in the differential diagnosis of pediatric patients with acute kidney insufficiency or chronic kidney disease of an unknown etiology. As far as we know, this is the first study regarding extrapulmonary sarcoidosis in children from Romania. Sarcoidosis is a systemic granulomatous disorder characterized by the accumulation of T lymphocytes, mononuclear phagocytes, and noncaseating granulomas in almost all affected tissues and organs. The clinical phenotypes vary from single-organ, sometimes self-limited, asymptomatic disease to multi-organ involvement with high-risk manifestations. The types of pediatric sarcoidosis are classified by age into two distinct forms: early onset sarcoidosis  and pediatric-onset adult-type sarcoidosis—preferentially involving the lung and mediastinum . Kidney 2 (Schwartz’s pediatric equation). He required admission to our pediatric nephrology department. He was hemodynamically stable, conscious, febrile, and in general distress. A follow-up evaluation revealed pancytopenia with a normal bone marrow assessment and no immunophenotyping abnormalities. Therefore, we concluded that the pancytopenia was in the liver damage. A negative carcinoembryonic antigen ruled out neoplastic disease. Moderate hepatic cytolysis and cholestasis were observed, with normal coagulation parameters. Hypercalcemia, low plasma parathyroid hormone levels, and metabolic acidosis were present, while urinalysis revealed hematuria, proteinuria, leukocyturia without hypercalciuria, and a negative urine culture. All of the differential diagnoses made upon admission to our department are summarized in We illustrate the case of a 10-year-old boy who presented for weight loss (4 kg in 12 months), fatigue, and feeling sick with an insidious onset in the last year. His past medical history includes multiple respiratory infections during early childhood and nonspecific enterocolitis one year before the current presentation. On this latter occasion, hypercalcemia, as well as elevated serum urea and creatinine levels, were detected but were considered secondary to dehydration, although they did not improve with adequate hydration. Hepatic cytolysis was also constantly present in the last year. Clinical examination revealed massive hepatosplenomegaly, pallor, and oliguria, while biologically, elevated serum creatinine was noted, corresponding to an eGFR of 25 mL/min/1.73 m2, requiring the initiation of hemodialysis on day 5 after admission. A history of drug ingestion that could cause acute interstitial nephritis was negative. Serologic tests for Epstein–Barr virus, cytomegalovirus, hepatitis C/B, human immunodeficiency virus, Toxoplasma gondii, and tuberculin skin reaction were negative. In the absence of specific immunological findings, a marked inflammatory syndrome or antibodies such as p-ANCA, c-ANCA or antinuclear antibodies, primary immunodeficiency, a connective tissue disease or vasculitis were excluded. Renal ultrasound evaluation was highly suggestive of nephrocalcinosis. After this preliminary evaluation, the diagnosis of advanced renal disease was made; therefore, the patient started chronic hemodialysis. Thoracic–abdominal computed tomography confirmed renal medullary calcifications, hepatosplenomegaly, hyperdense splenorenal and splenogastric masses as indicators of portal hypertension  was initiated, with positive results after 3 months of treatment, as presented in However, because of corticoid-related side effects (secondary Cushing syndrome), we decided to decrease the dose of prednisolone up to the maintenance dose of 0.25 mg/kg/day. Even though a clear histopathological diagnosis was made through hepatic punction, and renal involvement was almost certainly a consequence of sarcoidosis, this evolution prompted us to perform a renal biopsy. This showed non-granulomatous interstitial nephritis, nephrolithiasis, and interstitial fibrosis, as shown in Disease activity can be followed by angiotensin-converting enzyme (ACE) serum levels. We note an improvement of this parameter 3 months after cortisone therapy as well as an overall improvement of renal function, with dialysis discontinuation and hepatosplenomegaly diminution .Our evaluation concluded with the diagnosis of extrapulmonary pediatric-onset adult-type sarcoidosis with nephrocalcinosis, nephrolithiasis, non-granulomatous nephritis, granulomatous hepatitis, portal hypertension, hepatic insufficiency, splenic infarctions, and band keratopathy.Sarcoidosis is a disease characterized by the formation of nodules of inflammatory cells or noncaseating granulomas in any part of the body—most commonly in the lungs and lymph nodes. However, it can also affect the eyes, skin, heart, kidneys, central nervous system, or sense organs. Sarcoidosis, or Besnier-Boeck-Schaumann disease, was first identified by the English doctor Johnathan Hutchinson in 1877, and although more than a hundred years have passed since that first description, the disease still remains an enigma . The incSince 2008, with the Danish series, around 22 manuscripts on pediatric sarcoidosis have been published annually, compared to over 900 for adult sarcoidosis, with more than half being case reports . PediatrSarcoid granulomas represent an intensely interconnected network of immune cells, including macrophages, dendritic cells, T helper lymphocytes, T regulatory cells, and their mediators. After years of investigations and advances in medical science, the immunopathogenesis of sarcoidosis remains evasive. One or multiple antigens may trigger an exaggerated cell-mediated immune response resulting in granulomatous inflammation. These antigens, possible etiologic agents, vary from occupational or environmental factors to infectious agents, and, of course, genetic contribution may play an important role ,17,18. ABTNL2), annexin A11 (ANXA11), and angiotensin-converting enzyme variants, although the associations vary across populations [Special attention was directed to infectious agents, mostly mycobacteria and cutibacteria (in the past known as propionibacteria). Several studies showed their possible implications for developing sarcoidosis ,24. Mycoulations ,31. Drugulations .Sarcoidosis is diagnosed based on clinical and imaging manifestations as well as the histopathological detection of non-caseating granulomas at the level of the affected organs after excluding other diseases. No laboratory marker can be conclusive for the diagnosis of sarcoidosis, although serum angiotensin-converting enzyme is among the most commonly used diagnostic biomarkers for sarcoidosis; however, the test is nonspecific and lacks sensitivity . RegardiRegarding the clinical presentation, extrapulmonary manifestations were observed more frequently in symptomatic children and adolescents than in adults . Liver iThe reported prevalence of kidney involvement in sarcoidosis varies widely due to the study design and enrolled patient populations, but also due to the heterogeneity and asymptomatic nature of renal disease. Kidney lesions associated with sarcoidosis are represented by specific histological changes in the disease or by abnormal calcium metabolism, nephrolithiasis, and nephrocalcinosis. The renal manifestations in sarcoidosis are represented by hypercalcemia and hypercalciuria, nephrolithiasis and nephrocalcinosis, granulomatous or non-granulomatous interstitial nephritis, glomerular and tubular disease, and ureteral obstruction . A varieNephrocalcinosis is a result of chronic hypercalciuria and is a rare finding in sarcoidosis . It is pCoutant et al., retrospectively analyzed 11 children with renal granulomatous sarcoidosis confirmed by renal histology after a mean follow-up of 5.5 years; 3 patients developed end-stage renal failure, and 1 had chronic insufficiency after interruption of medical supervision and prednisone therapy . The evoDepending on the involvement, extrapulmonary sarcoidosis may benefit from local treatment, escalation of systemic treatment, or multimodal interventions . The majAdvanced sarcoidosis refers to manifestations of the disease with high mortality  and requires treatment in the stage of disease activity, with long-term maintenance therapy being controversial . TreatmeThe prognosis of the disease is generally good, with the exception of patients with severe cerebral deterioration or those with frequent relapses . The preSarcoidosis with extrapulmonary involvement in children is rare, which leads to a delayed diagnosis. In our case, renal sarcoidosis was diagnosed in advanced CKD, with severe GFR impairment at onset, offering unfavorable outcomes. Renal biopsy is mandatory in this case for a better understanding of the disease course and reassessment of the treatment. Sarcoidosis cannot necessarily be considered a benign nephrologic condition and should be considered in the differential diagnosis of pediatric patients with AKI/CKD of an unknown etiology. Oral or pulses of intravenous corticosteroids are the mainstay of the treatment of sarcoidosis in children. Relapses are frequently observed in the evolution of this pathology, mainly extrathoracic, so even in cases of initial remission, long-term follow-up is required. There are some clinical, radiological, and laboratory factors that determine the prognosis of the disease. The clinical phenotype of the disease can be presented with self-limited acute sarcoidosis and/or chronic, progressive multisystemic involvement. Laboratory factors are nonspecific and may not always be useful in clinical practice. New biomarkers would be useful for accurate diagnosis. New multicenter prospective studies are needed to shed light on this pathology, especially in children.


Idiopathic pulmonary fibrosis (IPF) is the most prevalent progressive idiopathic interstitial pneumonia. It has a poor prognosis  and typically presents in older individuals (mean age 74 years) [1]. Systemic hypertension is highly prevalent and the leading contributor to all-cause death and disability worldwide. Hypertension is a risk factor for common comorbidities of IPF including ischaemic heart disease, and >50% of individuals with IPF have systemic hypertension (defined as ≥130 mmHg systolic blood pressure (SBP) and/or ≥80 mmHg diastolic blood pressure (DBP) [2]). High blood pressure can lead to organ fibrosis or can be a consequence of artery stiffening due to fibrosis [3], and studies have implicated common pathways in both IPF and blood pressure regulation, such as transforming growth factor (TGF)-β signalling and the renin–angiotensin system [4]. The discovery of shared genetic associations, with sometimes different directions of effect, has implications for drug target discovery for idiopathic pulmonary fibrosis and systemic hypertension https://bit.ly/45IqbRv To the Editor:Idiopathic pulmonary fibrosis (IPF) is the most prevalent progressive idiopathic interstitial pneumonia. It has a poor prognosis  and typically presents in older individuals mean age 74 years) . Systemi4 years .−5 between IPF and either SBP or DBP; this was performed by identifying signals at p<10−5 in both datasets, and then performing colocalisation. We report signals with a posterior probability of >80% for a shared causal variant (coloc H4) as significant. Signals were mapped to a gene either using prior published evidence or using Open Targets Genetics (OTG). OTG utilises distance from gene, expression quantitative trait loci (QTL), splice QTL and functional annotation to suggest the most likely variant-to-gene mapping.We utilised the largest available genome-wide association studies (GWAS) of clinically defined IPF , and SBPMAD1L1, GOLPH3L/HORMAD1 and at 17q21.31) had the same direction of effect on risk of IPF and raised blood pressure (hypertension) and five  had opposite effects, with the allele associated with increased risk of IPF being associated with decreased blood pressure   or DBP . The genome-wide colocalisation analysis identified 18 regions of overlap, of which seven had a posterior probability of a shared causal variant >80% for IPF and blood pressure . In addi allele) .Our study identified shared genetic associations between IPF and blood pressure traits with genetic variants at some loci increasing the risk of both IPF and hypertension, while at other loci, the same genetic variants increased risk of one trait while being protective against the other.MAD1L1 (mitotic arrest deficient 1 like 1) encodes a component of the mitotic spindle assembly checkpoint and is a shared and consistent signal of association between IPF and hypertension. Common and rare genetic variants at the KIF15 (kinesin family member 15) gene [SPDL1 (spindle apparatus coiled-coil protein 1) [15) gene , and a rotein 1)  have alsotein 1) , 17.DEPTOR (DEP domain-containing mTOR-interacting protein) encodes a mammalian target of rapamycin (mTOR) inhibitor and decreased DEPTOR gene expression was associated with increased risk of IPF [k of IPF . Targetik of IPF . Our fin−8 will have partly mitigated this, but it is likely that more shared signals will be discovered as sample sizes increase. We have used published datasets that undertook adjustment for appropriate demographic covariates and restriction to individuals of European ancestry. Differences in age, sex and smoking history between the datasets may mean that we may have missed additional shared signals in our study. For example, where there may be interaction effects for either trait. None of the shared signals we highlight are associated with smoking.There was a significant difference in sample size for the GWAS used in our analyses and this will have affected the statistical power to detect signals in the smaller IPF dataset, meaning that there may be additional shared signals that we have not detected. Our genome-wide approach utilising a more lenient threshold than the commonly used genome-wide threshold of p<5×10We only report signals with a single shared causal variant for IPF and blood pressure. It is plausible that distinct causal variants could exert their effect through the same nearby gene or different genes in the same molecular pathway; however, this study was not designed to detect such effects. It is likely that genetic effects may be tissue-specific and that different regulatory pathways may be involved in the expression of the same gene in different tissues.vice versa), it is important to understand the mechanisms of such shared pathways and to ensure that genetically indicated potential adverse effects are proactively monitored in future clinical trials for new drugs.Our findings provide support that IPF and systemic hypertension have shared mechanisms which may promote both fibrosis and hypertension, as well as others where there are opposite directions of effect. Given the frequency of hypertension and ischaemic heart disease as comorbidities in IPF, and the effect of potential antifibrotics on blood pressure (and


Definition of the microbiology of pleural space infection has been challenging due to the poor yield of conventional culture, a challenge that has led to recommendations for broad spectrum antimicrobial therapy for these infections. Since August 2019, we have offered a 16S ribosomal RNA gene PCR assay followed by Sanger or next generation sequencing, or reported as negative, depending on the PCR signal strength  in clinical practice at Mayo Clinic.Here, we describe the microbiologic and clinical findings of 57 pleural fluids testing positive by 16S rRNA gene PCR/sequencing from August 2020 to January 2023. Demographic data and clinical features, and results of pleural fluid and blood cultures were recorded. Analysis was performed using SAS software version 9.4 (SAS Inc).Fusobacterium nucleatum group and/or Streptococcus intermedius were detected in 31/57 (54%) cases [including 28/48 (58%) in the subset with community-acquired infection], with additional facultative and/or anaerobic species in various combinations also found in 17/31 (55%) of these cases.Median patient age was 63 years (range 5-83 years) and 75% were men. Pleural space infection was community-acquired in 84%. The most frequent symptom was dyspnea (80%), followed by chest pain (52%). 89% of patients had received antibiotics in the two weeks preceding specimen collection. Radiological consolidation was observed in 63% and pleural fluid was loculated in 58%. Pleural fluid and blood cultures were positive in 32 and 11%, respectively. The microbiology found with the sequencing assay is shown in the table. 23/57 (40%) detections were polymicrobial. Fusobacterium nucleatum group and/or Streptococcus intermedius, with or without other normal microbiota, a grouping for which we propose the term FuSion .Based on analysis of pleural fluid using a targeted metagenomic sequencing assay in clinical practice, the most frequent organism profile involved in community-acquired pleural space infection is a combination of Eva Carmona, MD, boehringer ingelheim: Advisor/Consultant Øyvind Kommedal, n/a, Pathogenomix inc.: Advisor/Consultant|Pathogenomix inc.: Stocks/Bonds Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant


BackgroundHealthcare-related artificial intelligence (AI) is developing. The capacity of the system to carry out sophisticated cognitive processes, such as problem-solving, decision-making, reasoning, and perceiving, is referred to as higher cognitive thinking in AI. This kind of thinking requires more than just processing facts; it also entails comprehending and working with abstract ideas, evaluating and applying data relevant to the context, and producing new insights based on prior learning and experience. ChatGPT is an artificial intelligence-based conversational software that can engage with people to answer questions and uses natural language processing models. The platform has created a worldwide buzz and keeps setting an ongoing trend in solving many complex problems in various dimensions. Nevertheless, ChatGPT's capacity to correctly respond to queries requiring higher-level thinking in medical biochemistry has not yet been investigated. So, this research aimed to evaluate ChatGPT's aptitude for responding to higher-order questions on medical biochemistry.ObjectiveIn this study, our objective was to determine whether ChatGPT can address higher-order problems related to medical biochemistry.MethodsThis cross-sectional study was done online by conversing with the current version of ChatGPT . It was presented with 200 medical biochemistry reasoning questions that require higher-order thinking. These questions were randomly picked from the institution's question bank and classified according to the Competency-Based Medical Education (CBME) curriculum's competency modules. The responses were collected and archived for subsequent research. Two expert biochemistry academicians examined the replies on a zero to five scale. The score's accuracy was determined by a one-sample Wilcoxon signed rank test using hypothetical values.ResultThe AI software answered 200 questions requiring higher-order thinking with a median score of 4.0 . Using a single sample Wilcoxon signed rank test, the result was less than the hypothetical maximum of five (p=0.001) and comparable to four (p=0.16). There was no difference in the replies to questions from different CBME modules in medical biochemistry . The inter-rater reliability of the scores scored by two biochemistry faculty members was outstanding (ICC=0.926 (95% CI: 0.814-0.971); F=19; p=0.001)ConclusionThe results of this research indicate that ChatGPT has the potential to be a successful tool for answering questions requiring higher-order thinking in medical biochemistry, with a median score of four out of five. However, continuous training and development with data of recent advances are essential to improve performance and make it functional for the ever-growing field of academic medical usage. Artificial intelligence (AI) has various definitions. Still, none of them can likely adequately explain this complicated phenomenon, which is currently the focus of numerous research efforts across many scientific fields in addition to the realm of information technologies. Machines with artificial intelligence can recognize a problem and develop a workable solution using methods similar to human cognitive and psychological processes. The system learns how to accurately forecast a variable's value, classify a set of data, or carry out other challenging tasks using artificial intelligence techniques ,2.In recent years, numerous initiatives have been taken to use artificial intelligence and machine learning in molecular medicine, biochemistry, and cell biology ,4. Many AI systems' capacity for advanced cognitive functions, including problem-solving, decision-making, reasoning, and perceiving, is called higher cognitive thinking. This thinking requires more than just processing facts; it also entails comprehending and working with abstract ideas, interpreting and applying relevant information, and producing new insights based on prior learning and experience . HoweverIn the fields of biochemistry and laboratory medicine, AI's capacity to solve higher-order reasoning-type issues depends on the difficulty of the questions being asked and the quality of the training data the AI system has been presented with. AI systems can instantly and accurately respond to simple or uncomplicated inquiries . For insOne such AI-based conversational tool, ChatGPT, is being tested for biomedical writing and can produce replies that resemble human answers . For resStudy setting and ethical considerationThis cross-sectional study was conducted at the biochemistry department of Dr. B.C. Roy Multi-Speciality Medical Research Centre, Indian Institute of Technology Kharagpur, over the second and third week of March 2023. The information used in this research was gathered using the recent version of Chat GPT , presently an open-source free online tool for registered users. For data collection, we used laptops with institutional Wi-Fi broadband internet connection. There are no human research subjects in this study. As a result, the research is exempted from institutional ethical review under current norms.QuestionsAs per the competencies of biochemistry in the Competency-Based Medical Education (CBME) curriculum, as designed by the National Medical Commission for Indian Medical Graduates, we divided the entire curriculum into 11 sections. This topic-wise competency mapping is shown in Table Using the department's question bank, which is a compilation of first and second semester questions from various medical universities across India, we picked a total of 200 questions at random, covering the entire curriculum. Among them, 100 questions were reasoning or justification type of questions, which are typically set in first professional Bachelor of Medicine and Bachelor of Surgery (MBBS) examinations without any alternative options, and these are considered to be of higher order and an in-depth understanding of the topic is necessary to provide a solution. Instead of merely memorizing information, it focuses on fundamental ideas and principles. For instance, asking someone to apply a concept to a new circumstance requires analysis and synthesis of information as opposed in asking them to recollect a structure or description . We alsoData collectionThe process of gathering information occurred between March 14 and March 16, 2023. The questions were utilized to initiate the dialogue with ChatGPT. The response to reasoning type of questions given by the software was transcribed onto a notepad and stored on the computer to be examined later. Regarding the MCQs, we asked Chat GPT to provide the reasoning for choosing the answers. The first response was taken as final, and the option of "regenerate response" was not used. Thereafter the collected notes were printed out for evaluation. Scoring was done by two assessors on a scale of zero to five, with zero being incorrect and five being fully correct, based on a pre-selected answer key.Statistical analysisThe data were expressed as number, mean, median, standard deviation, and first and third quartiles using descriptive statistical tests. The Kolmogorov-Smirnov test was performed with the dataset, which showed the distribution was not normal. We conducted a one-sample Wilcoxon signed rank test using hypothetical anticipated values to assess the response's correctness. The intraclass correlation coefficient was used to evaluate the score between the two raters (ICC). For all of the statistical analyses, we utilized SPSS version 21 . A p-value of less than 0.05 was regarded as statistically significant. A brief outline of the study design has been highlighted in Figure The AI software answered a total of 200 questions requiring higher-order thinking with a mean score of 3.98 (± 0.70) and a median score of 4.0. . The overall and system-wise scores of the responses are shown in Table Using a single sample Wilcoxon signed rank test, the result was less than the hypothetical maximum of five (p=0.001) and comparable to four (p=0.16). The median score of BI 1 was near to five, and the median score of modules BI 10 and BI 11 was below four . There was no difference in the replies to questions from different CBME modules in medical biochemistry . The p-values from the post hoc test were not presented because there was no discernible difference between the two groups. The inter-rater reliability of the scores scored by two biochemistry faculty members was outstanding. The ICC was 0.926 with a 95% confidence interval of 0.814 to 0.971 . A few examples of Chat GPT responses are shown in the appendix. We tested ChatGPT's ability to solve complex biochemistry questions by giving it 200 random questions. Based on our findings, ChatGPT's responses scored at least four out of five marks on all the modules except the last two modules, where the score was lower. Numerous past investigations have evaluated the potential of ChatGPT for medical education purposes. The observation is similar to a recent study conducted by Sinha et al. demonstrating the utility of ChatGPT for solving higher-order problems in pathology . The stuOn the other hand, Huh's study discovered that ChatGPT's performance in parasitology is still lacking compared to that of a Korean student . The stuWe recommend that medical schools and colleges should not impose limitations on the utilization of AI. Instead, the students should be equipped with the necessary skills to use them optimally. This is of utmost importance that when a student is trying to solve MCQs, they can readily get the answer from ChatGPT with proper reasoning. Simply pasting an MCQ the same into an online search engine may not give any useful response. It is imperative to ensure that future AI systems are designed, developed, and validated meticulously to provide medical students with reliable and precise information. As AI continues to advance, there is a need for further development in health-related information to increase its potential to be incorporated into education and healthcare systems . It is aLimitationsThere are various limitations to this study. Firstly, we employed a scoring approach that ranged from zero to five. Despite preparing the answer keys beforehand, there may still have been a subjective bias in the assessment that was beyond our control. The questions used in our study were sourced from our question bank, and other institutions may have different questions. Therefore, future studies may need to be conducted multicentrically for a more generalizable outcome. Furthermore, even a slight modification to a question may result in a different response from ChatGPT, so this should be considered in future investigations.It can be concluded that ChatGPT helps in seeking answers for higher-order reasoning questions in medical biochemistry. The study showed a median score of four out of five in solving the questions with better performance in explaining traditional concepts rather than justifying recent advances due to its limitation in training databases in the constantly evolving medical research in cancer biochemistry, immunology, and clinical investigations. It can be an excellent tool for solving multiple-choice questions and getting the proper reasoning behind the solution. Although such cognitive ability in AI can benefit students and academicians seeking quick and functional responses to their inquiries, considering the evolution of AI programs worldwide, testing their capabilities in future studies in various medical disciplines is imperative.