Undiagnosed malnutrition is a significant problem in high-income countries, which can reduce the quality of life of many individuals, particularly of older adults. Moreover, it can also inflate the costs of existing health care systems because of the many metabolic complications that it can cause.The current methods for assessing malnutrition can be cumbersome. A trained practitioner must be present to conduct an assessment, or patients must travel to facilities with specialized equipment to obtain their measurements. Therefore, digital health care is a possible way of closing this gap as it is rapidly gaining traction as a scalable means of improving efficiency in the health care system. It allows for the remote monitoring of nutritional status without requiring the physical presence of practitioners or the use of advanced medical equipment. As such, there is an increasing interest in expanding the range of digital applications to facilitate remote monitoring and management of health issues.In this study, we discuss the feasibility of a novel digital remote method for diagnosing malnutrition using facial morphometrics. Many malnutrition screening assessments include subjective assessments of the head and the face. Facial appearance is often used by clinicians as the first point of qualitative indication of health status. Hence, there may be merit in quantifying these subtle but observable changes using facial morphometrics. Modern advancements in artificial intelligence, data science, sensors, and computing technologies allow facial features to be accurately digitized, which could potentially allow these previously intuitive assessments to be quantified.This study aims to stimulate further discussion and discourse on how this emerging technology can be used to provide real-time access to nutritional status. The use of facial morphometrics extends the use of currently available technology and may provide a scalable, easily deployable solution for nutritional status to be monitored in real time. This will enable clinicians and dietitians to keep track of patients remotely and provide the necessary intervention measures as required, as well as providing health care institutions and policy makers with essential information that can be used to inform and enable targeted public health approaches within affected populations. Malnutrition in older adults in many high-income countries is an unrecognized condition that is increasing in prevalence as the population ages . The impanorexia of aging, has severe implications, resulting in a significantly poor quality of life [Malnutrition in older adults, also coined as  of life ,4. The l of life ,6. Malnu of life . Malnutr of life .Malnutrition has been found to lead to sarcopenia, and sarcopenia may aggravate malnutrition, leading to the aggravation of morbidity and mortality ,10. SarcApart from physiological and metabolic implications, malnutrition and sarcopenia in older adults have been widely established to have financial implications and are burdens to health care resources . PatientHowever, it is difficult to detect subclinical malnutrition because of the absence of clear indicators. This could lead to a lack of identifying poor nutritional practices among older adults, as malnutrition does not often present with immediate problems. Therefore, better screening options for the diagnosis of subclinical malnutrition must be recognized and identified ,26. ThisIt is imperative that malnutrition and the prognostic value of treatment options to manage malnutrition be identified. The face is often used by health practitioners to make qualitative assessments of health and nutritional status. Modern advancements in sensor and computing technologies enable features in the face to be accurately digitized and could potentially allow these previously intuitive assessments to be quantified. In this paper, we discuss the feasibility of a novel digital remote method for diagnosing malnutrition using facial morphometrics.Until 2016, there were no universally accepted screening tools or criteria used as the gold standard to identify malnutrition risk . The GloOne of the most widely used traditional methods for assessing malnutrition is the subjective global assessment tool. It is an integrated tool that assesses nutritional status based on patients’ past records and physical examinations, and subsequently classifies patients into three categories: well-nourished, moderately malnourished, or severely malnourished . AnthropDigital health care is one such approach that allows for malnutrition and nutritional status to be monitored remotely without requiring the physical presence of practitioners or advanced equipment. As we enter the Fourth Industrial Revolution, the widespread availability and improvements in data science and infocommunication (integration of information and telecommunication technology sectors) technologies have rapidly expanded the vast range of available services and applications in the health care sector -39. DigiTelemonitoring is the real-time monitoring of patients using mobile technologies to conduct routine medical tests and communicate results to health care workers for evaluation . This caTelemonitoring is also considered an efficient use of health care resources -44,46,48Given the many benefits that telemonitoring has to offer, it is critical that new applications are developed to broaden the scope of conditions to which telemonitoring can be applied. In both high- and low-income nations, smartphones have been used as platforms for several aspects of life, such as identity verification and personal banking, and are increasingly being adopted for use in the capture, management, and transmission of personal health data -54. ThesA relatively unexplored method that may offer predictive capabilities in the area of malnutrition screening is the application of facial morphometrics. Facial morphometrics is the study of the contours and structures of faces using geometric mapping tools that allow landmarks and features to be identified . AlthougThe newly integrated depth sensors in personal smart devices may present a novel solution that may be beneficial for the remote monitoring of malnutrition. These depth sensors capture 3D depth data of objects, enabling the use of applications such as augmented reality–based applications, as well as the generation of 3D point clouds for 3D modeling . Most reWith depth cameras becoming the mainstay in smartphones beyond the iPhone, there may be potential for developing telemonitoring applications that involve the extraction and correlation of facial morphological features with health indices. Typical facial landmarks include the eyes, nose, chin, cheekbones, and the overall shape of the face. The extraction and analysis of features such as geodesic distances or ratios between these landmarks can serve as informative discriminatory tools in the quantification of shape and variation .These quantified features could serve as proxy markers for the nutritional status of the human body. This has been demonstrated in several studies, which have observed correlations between superficial tissues that affect the overall morphology of the face such as the zygomatic fat pads and masseter and temporalis muscles with nutritional status and whole-body SMM -83. The The use of facial morphometrics in the field of health sciences is not new. However, most of the research has primarily been conducted in the fields of aesthetics, orthodontics, and prosthetics. There have been attempts to explore the correlation between facial features and nutrition-focused indices such as BMI and waist-to-hip ratio -91. EarlWith these capabilities in mind, this paper further discusses the relevance of certain facial features that have the potential to provide diagnostic value in identifying changes in SMM and overall nutritional status.With imaging tools capable of deriving facial morphology accurately, both direct and indirect tissue losses can potentially be captured as additional features for analysis. Direct measurements of tissue loss can include the quantification of protrusions and depressions that form from tissue atrophy, whereas indirect measurements include features that become more prominent when there is a loss of fat or muscle tissue, such as certain bone processes and ridges, or the angulation of the jawline ,100,103.The degree of bone prominence is important in determining nutritional status ,105. PhyTo discuss potential features, we segment the face into three regions: upper face, midface, and lower face .The upper face comprises the regions between both temples, including the eyes and the surrounding periorbital region. The primary superficial tissues that contribute to craniofacial morphology in this region include the temporalis muscle, periorbital fat pads, and bones that make up the orbital rim ,106,107.The temporalis muscle, as well as the superficial and deep temporal fat pads, contribute to most of the volume in the temple regions . As it iIn the case of atrophy of the underlying fat pads and the temporalis muscle, this region could appear sunken, thus increasing the prominence of the temporal orbital rim . The froThe morphology of the periorbital region also changes significantly in the case of tissue loss and is largely determined by the volume of the periorbital fat pads. These comprise the temporal fat pad, the preaponeurotic fat pad, the nasal fat pads, and the central fat pad  Figure . In caseCertain confounders may arise because of the natural changes around the eye regions, which may complicate the identification of malnutrition. Age contributes to a decrease in brow projection from the superior orbital rim . YouthfuIt has been well established that the morphology around the periorbital fat pads is affected by many factors because of aging ,116,117.The midface comprises the regions bound by the cheekbones as the upper boundary and the lips as the lower boundary. Key tissue groups that affect the morphology of this region include the masseter muscle, the various zygomatic-malar fat pads, and the zygomatic bone ,107.The masseter muscle is a masticatory muscle located over the zygomatic bone, running from the temporal bone to the lower jaw . Given tSimilar to the usability of the temporalis muscle as a surrogate marker for SMM, the masseter muscle has also been found to be correlated with whole-body SMM and the psoas, limb, and trunk muscles ,126,129.Given the masseter muscle’s key function as one of the primary muscles of mastication, its strength has also been found to be correlated with the chewing ability ,130,133.There are several fat compartments in the zygomatic-malar region of the face. The deep midface compartments include the medial and lateral suborbicularis oculi fat, the medial portion of the buccal fat pad, and the deep medial cheek fat. Superficial compartments include the nasolabial and middle cheek superficial fat compartments  107,116116. The Subcutaneous buccal fat pads are also a significant area of aging-associated fat loss . The losThe lower face comprises the mandible and the surrounding tissues of the lower cheeks, chin, and neck. These structures contribute significantly to the overall shape of the face and are often easily perceptible, except in cases of excess facial hair. As such, the lower face is a key point of reference in many facial morphological studies and BMI prediction algorithms ,135,136.The lower mandible contributes to the morphology of the lower face and accentuates the jawline. Aesthetic surgeons refer to a youthful jawline as one characterized by a straight line from the chin to the mandibular angle ,138. ExcThe jawline can be a potentially important feature in predicting malnutrition or sarcopenia. In a study exploring the relationship between face shape and waist-to-hip ratio, Mayer et al  showed tAlthough subcutaneous fat affects the overall shape of the jawline, aging is another important factor that changes the overall morphology of this facial feature ,138-140.Facial morphology technology provides several exciting opportunities as a diagnostic tool for malnutrition and body composition. However, there remain important challenges that need to be overcome.Developing a comprehensive database that accounts for potential confounders and isolating the degree of correlation between various facial morphological features and overall SMM and nutritional status is necessary. Many factors can influence facial variations outside of weight and SMM, such as gender, ethnicity, age, and medical conditions ,142,143.Facial morphology holds potential as a diagnostic tool in the context of malnutrition on the premise that SMM is correlated with facial tissues. However, this correlation may not always be consistent, particularly for complex medical conditions. Certain diseases such as critical illness myopathy have been noted to affect muscles in the body differently, resulting in significant muscle loss in the limbs and trunk while sparing the masseter and other craniofacial muscles . ControlThere can also be disproportionate tissue loss in specific parts of the face, which may confound the degree of importance of the respective facial regions. For example, patients undergoing highly active antiretroviral therapy for HIV infections or patients with connective tissue diseases such as panniculitis are known to develop asymmetrical and dynamic facial lipoatrophy . This foThe use of facial morphology as a diagnostic tool may also require additional considerations because of aesthetic variables. The presence of facial hair, such as beards and mustaches, may impair the ability of features derived from the lower half of the face to be used in the analysis. Cosmetic or reconstructive surgery used for the concealment of facial anomalies or to improve one’s appearance is also becoming relatively common in many countries . These cCertain ethical and societal considerations may need to be reviewed to increase the adoption of facial morphometrics as a tool in public health. The face is an important and sensitive identifiable feature, and any application incorporating digital 3D scans of the face will need to ensure that patient privacy and data security are not compromised. The development of digital health applications using personal smartphones may also widen the digital divide and potentially alienate certain segments of the population . PopulatUndiagnosed malnutrition is becoming a significant problem that can reduce the quality of life of many individuals and inflate the costs of existing health care systems because of the many metabolic complications it can cause. Many high-income countries are faced with a rapidly aging population that may lead to poor food intake, disease etiology, and improper access to food, which may give rise to an increase in the prevalence of malnutrition. Given the need for specialized equipment and trained practitioners for the diagnosis of malnutrition, the development of simpler and more scalable methods for assessing malnutrition has been a long-standing challenge for public health.Recent advancements in artificial intelligence, computing, data science, and infocommunication technologies present a wealth of opportunities that will shape and revolutionize the landscape of health care. This paper aims to stimulate further discussion and discourse on how adopting this emerging technology could provide real-time access to nutritional status. We explored the feasibility of a novel, noninvasive approach to tackling malnutrition in the community. The use of facial morphometrics extends the use of currently available technology, providing a scalable and easily deployable method for the real-time monitoring of nutritional status. Clinicians and dietitians will be able to keep track of patients remotely and provide the necessary intervention measures as required. Finally, it will furnish health care institutions and policy makers with essential information that can facilitate necessary interventions targeted at affected populations.


Cerebral arteriovenous malformations (AVMs) are rare vascular lesions that rupture at an approximate annual rate of 2–4% . Annual The complications of AVM GKRS are mainly two-fold. Firstly, patients remain at risk of haemorrhage during the latency period between GKRS and nidus obliteration. The relative risk of AVM haemorrhage during this latency period versus the natural history of an untreated AVM remains a topic of debate , 28. SecA previous systematic review and meta-analysis quantified haemorrhage risk and obliteration rate following AVM stereotactic radiosurgery (SRS), but it had limitations . The revAlongside imaging advancements and increasingly sophisticated radiation delivery systems since the inception of GKRS, there has also been an increasing knowledge base on AVM obliteration, RICs and post-GKRS ICH . Whilst We performed this systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines Appendix  53]. Th. Th53]. We performed a comprehensive literature search Appendix  using OVWe sought randomised trials and observational cohort studies, published in English in peer-reviewed journals reporting 20 + adult (18 +) patients with AVMs, diagnosed by MRI or histopathological examination, treated with single-session GKRS. We included studies describing all the following patient and AVM characteristics: (1) median (or mean) margin dose, (2) AVM volume (or maximum nidus diameter), (3) clinical presentation, (4) AVM Spetzler-Martin grades. We included studies reporting all the following clinical and radiological outcomes, with a minimum follow-up period of 12 months following GKRS: (1) complete nidus obliteration rate (angiography or angiography/MRI-confirmed), (2) post-GKRS ICH, (3) RICs or adverse radiation effects (ARE).Two investigators (MC and AV) independently screened all titles and abstracts for eligibility. The full text of eligible studies was reviewed for inclusion. CSH, CT and PG acted as mediators in cases of disagreement. If multiple studies with overlapping data from a single institution were published, the study reporting the largest sample size and/or the most recent study were prioritised Appendix  and 4.Data extraction was performed independently by two authors (MC and AV) from each unique study cohort undergoing GKRS to ensure consistent extraction of patient, AVM and treatment characteristics. We extracted the following outcomes: complete nidus obliteration rate, ICH, radiologic evidence of RIC, symptomatic RIC events (transient or permanent), radionecrosis, cyst formation, radiation-induced neoplasm, seizures (new or worsening), deaths attributed to AVM/GKRS (case-related) and all-cause death rate.We used outcomes as described per patient. The proportion successfully obliterated was calculated as a proportion of all patients with available radiological follow-up and was defined as the presence of angiographically demonstrated complete obliteration and/or angiography-or-MRI confirmed obliteration. Post-GKRS ICH was defined as any AVM-related haemorrhage detected through appropriate follow-up radiological imaging. RICs were classified as follows: (1) total radiologic RIC: any MRI evidence of peri-nidal T2-weighted hyperintensities after GKRS; (2) transient symptomatic RIC: radiologic RIC which correlated with new or worsening neurological symptoms, typically headache, seizure or focal neurological deficit, which resolved by the end of study follow-up; and (3) permanent RIC: symptomatic RIC without full recovery to pre-GKRS neurological baseline at the end of study follow-up.Two authors (MC and AV) assessed the risk of bias of each cohort as serious, moderate, low, or unclear risk according to the following domains of the Cochrane ROBINS-I tool : (1) conWe quantified the distribution of cohort-level characteristics with descriptive analyses. We calculated the incidence of clinical outcomes as a proportion of total patients treated with GKRS. We quantified the occurrence of haemorrhage and fatality at any time after GKRS either during the total person-years of follow-up stated or by multiplying the median (or mean) follow-up duration by total number of patients treated. We calculated annual incidence rates for haemorrhage and fatality, using Poisson distributions.We aimed to perform the following subgroup analyses: (1) clinical and obliteration outcomes reported in studies stratifying by AVM Spetzler-Martin grades : I–II, IA meta-analysis was performed for all primary outcomes. DerSimonian-Laird random effects models were used for all summary effect estimates with a Freeman-Tukey double arcsine transformation , 25.I2 [Heterogeneity was assessed using the quantity I2 . HeterogI2  and leav3), deep location (%), eloquent location (%), deep venous drainage (%).Moderator analysis was performed using meta-regression techniques to determine association of patient, AVM and treatment characteristics with clinical and radiological outcomes. Patients were stratified by the following pre-specified variables: age, sex, study mid-year, median margin treatment dose (Gy), median AVM volume  was used for all statistical analyses. For all statistical tests, After screening 3402 publications, 166 full-text studies were reviewed with 34 studies included in final analysis  receiving GKRS, including a total of 8673 patients with 41,544 patient-years of follow-up, the median cohort-level patient characteristics were: population size 182 patients, age at initial treatment 35 years, 53% male, follow-up duration 60 months, 49% presented with ICH, 71% AVMs were lobar, 20% deep and 72% in eloquent locations (Table Thirty-three 97%) studies were from single centres and one (3%) was multi-centre . Annual case-fatality rate was 0.46% (95% CI 0.37–0.57). Annual all-cause fatality rate (AVM/non-AVM related) was 0.67% (95% CI 0.58–0.76). All annual incidence of outcomes were calculated per 100 person-years of follow-up over the duration of follow-up described in included studies.Complete nidus obliteration, confirmed on angiography imaging, was achieved in 56.7% 3092/5450) of patients in 21 cohorts, at the end of follow-up after single-stage Gamma knife treatment 50 of pat, 58, 65.I2 = 63%)   .On pooled analysis, post-GKRS ICH rate was 6.1%  Fig. ; and casI2 = 95%) of AVMs were confirmed obliterated on angiography imaging and 69.7%  of AVMs were confirmed obliterated on either angiography-or-MRI imaging . One potential outlier was identified within the transient symptomatic RIC pooled analysis [I2 = 66%).Sensitivity analysis identified one outlier within the haemorrhage pooled analysis . Excludianalysis . Excludip = 0.005) and eloquent location (p = 0.026) were associated with higher haemorrhage rates. The chance of complete AVM obliteration was higher in more recent cohorts (p = 0.021) with younger patients (p = 0.018) or AVMs with deep venous drainage (p = 0.035). A higher risk of both transient and permanent symptomatic RIC was associated with cohorts with higher proportions of AVMs in deep locations (p = 0.002 and 0.005). Appendix Cohorts with higher proportions of AVMs with strictly deep venous drainage , overall reported obliteration rate was 80%. Of 888 SM I/II AVMs (72% SM II) in our study, 74% achieved obliteration. Both findings highlight that appropriately selected, low-grade AVMs can expect a significant chance of successful outcome.On behalf of the International Stereotactic Radiosurgery Society (ISRS), a systematic review was performed to establish SRS practice guidelines for SM I/II AVMs . Of 1102Spetzler-Martin grade III AVMs are angioarchitecturally heterogeneous lesions, straddling the boundary between low grade (I–II) AVMs, which favour intervention with microsurgical resection, and high grade (IV–V) AVMs, which favour conservative management. Our findings suggest grade III AVMs experience a reasonable risk-to-benefit profile following GKRS; of 505 AVMs, 69% achieved obliteration.Whilst our pooled and SM-stratified obliteration values entice direct comparisons with obliteration outcomes following surgical resection, caution is warranted as patient selection for GKRS versus surgical resection is undoubtedly to have been influenced by individual patient and AVM features which were not available for further detailed analysis.3 and dose 20 Gy) underwent single-stage GKRS with ≥ 2 years of follow-up, with reported symptomatic and permanent RIC rates of 7.1% and 3%, respectively. Increased AVM volume, 12-Gy volume, higher margin dose and deep location were associated with a higher rate of developing symptomatic RICs [p = 0.45 and 0.43) or permanent RICs (p = 0.17 and 0.19). We believe that this may be due to lack of individual patient data, differences in patient selection and variations in GKRS treatment protocols among included studies; all of which precluded a sufficiently rigorous analysis of the relationship among study nidus volume, margin dose, and symptomatic RIC occurrence. Furthermore, this dose-volume relationship has been further refined to specify the ‘12-Gray volume’, the volume of brain tissue receiving radiation dose of 12 Gy or more, which is strongly correlated with the risk of RICs [Previous studies have reported on patient, AVM and treatment factors associated with the development of RICs, of which, increasing radiation dose and irradiated AVM volume, have been well established , 17, 40. of RICs . Interes of RICs  referred of RICs , 55.p = 0.002) and permanent symptomatic RICs (p = 0.005) and is reflected in the RBAS grading system where deep location confers a higher risk feature associated with poorer outcome post-GKRS [One AVM angioarchitectural feature consistently reported to be associated with the development of symptomatic RICs is deep location, more specifically, the brainstem , 22, 61.ost-GKRS .The relative risk of AVM haemorrhage during the latency period between radiosurgery and obliteration versus an untreated AVM’s natural history remains a topic of debate . In compFurther, in our analysis, we were unable to determine whether pre-GKRS AVM rupture status, yielded a significantly lower or higher post-GKRS haemorrhage risk as (1) the majority of studies did not stratify haemorrhage rate by rupture status, and (2) we did not have access to individual patient data to perform rigorous sub-group analysis within individual cohorts. Yen et al.  evaluateCyst formation (CF) is an uncommon complication following SRS. In our review, 15 studies totalling 3446 patients reported a pooled incidence of 2%. This is approximated by the only comprehensive analysis of CF following GKRS by Ilyas et al., reporting a pooled CF rate of 2.9% in 2562 patients following GKRS, with a mean latency period of 6.5 years from time of GKRS to cyst detection . A shortThe strengths of this review include adherence to PRISMA guidelines, its pre-specified protocol and formal validated risk-of-bias assessment. Studies included in this review generally followed a similar AVM GKRS treatment and follow-up protocol, of which the median length of follow-up across all studies was approximately 5 years.I2 values found in cohorts reporting obliteration outcomes. We are aware that certain studies may have been excluded whilst strictly adhering to our inclusion criteria which required outcomes data on obliteration, haemorrhage and RICs cumulatively or excluded solely due to English language restriction  in this review reported an aggregated angiography or MRI confirmed obliteration rate, some in addition or as an alternative to an angiography-only confirmed obliteration rate. Whilst MR-imaging has been shown to exhibit 77–85% sensitivity and 89–95% specificity for AVM obliteration detection , 63, theIn this review, we obtained aggregate patient, AVM and outcome data with a tendency for studies to report on the whole patient cohort with limited further sub-group analysis. Very few studies stratified outcomes by individual patient and AVM features, e.g. by location, AVM volume, margin dose or SM grade, which have been well-established to affect radiosurgical outcomes. Further, the reporting of a clinically heterogeneous cohort of patients with AVMs is likely to contribute to heterogenous pooled estimates of outcomes, as illustrated in our findings. Interpreting these summary outcome estimates, without robust sub-group analysis can mitigate the generalisability of our findings for individual patients and future AVM treatment decision-making.Standardised prospective multi-centre recording of patient, AVM and treatment characteristics and reporting of outcomes is needed to be certain of individual AVM risks and benefit prediction. Stratifying clinical outcomes further by AVM and pre-specified treatment subgroups will contribute to the selection of a more homogenous set of AVMs from which meaningful comparisons of outcomes can be made and valid conclusions can be drawn.Gamma knife radiosurgery is a safe and effective treatment option for cerebral AVMs. Appropriately selected patients can expect a significant chance of successful obliteration coupled with minimal risks of haemorrhage and radiation-induced complications. Future studies would be strengthened by attempting to report on a homogenous set of study participants, in terms of pre-specified patient, AVM angioarchitectural or treatment parameters, which would allow for a more conclusive risk: benefit profile of AVM GKRS to be established.Supplementary file1 (PDF 764 kb)Below is the link to the electronic supplementary material.


The 10 known BRICHOS domain-containing proteins in humans have been linked to anunusually long list of pathologies, including cancer, obesity and two amyloid-likediseases. BRICHOS domains themselves have been described as intramolecular chaperonesthat act to prevent amyloid-like aggregation of their proteins' mature polypeptides.Using structural comparison of coevolution-based AlphaFold models and sequenceconservation, we identified the Out at First (OAF) protein as a new member of theBRICHOS family in humans. OAF is an experimentally uncharacterized protein that has beenproposed as a candidate biomarker for clinical management of coronavirus disease 2019infections. Our analysis revealed how structural comparison of AlphaFold models candiscover remote homology relationships and lead to a better understanding of BRICHOSdomain molecular mechanism.Bioinformatics Advances online. The 10 human BRICHOS domain-containing proteins are divided among five subfamilies:proSP-C/SFTPC (one member), ITM/BRI , BRICD5 (one member),Gastrokines  and Tenomodulin/Chondromodulin (TNMD and CHM1/LECT1). Formost, their precursors consist of three parts: an N-terminal transmembrane region, part ofeither a signal peptide for secretion or a signal-anchor for type II membrane proteins,followed by a long BRICHOS domain and a C-terminal shorter mature polypeptide generated byproteolytic cleavage. BRICHOS domain-containing proteins have been characterized aspre-pro-proteins, which once secreted, undergo proteolytic processing, usually by proproteinconvertases, releasing their mature form . These eMature forms of some invertebrate members of the BRICHOS domain-containing family, such asarenicin, alvinellacin, nicomicin and capitellacin, have been characterized as antimicrobialpolypeptides able to aggregate on lipid membranes and which exhibit detergent-likeproperties  or else convergence, we next investigated amino acid sequence similaritiesbetween OAF and the BRICHOS domain sequences. For this, we performed a sequenceconservation analysis using the HHpred profile-to-profile comparison tool .OAF shows five conserved features commonly found in BRICHOS family members ; (iv) aStatistical significance of sequence and structural comparisons, and the presence in OAFof five features conserved among the BRICHOS family, are sufficient to infer that OAF isan 11th and previously unanticipated member of the human BRICHOS family.Oaf was originally described in Drosophila, where itsfunction was related to neuronal development and hatching (Caenorhabditiselegans (Pfam entry: PF14941) (http://www.mousephenotype.org/data/genes/MGI:94852).Oaf inDrosophila nephrocytes reduces albumin endocytosis  has been recently identified in OAF, anddescribed as a putative cause of a musculoskeletal and neurological developmental disorder. Its mutDrosophilamelanogaster Oaf), indicative of functional conservation.OAF’s predicted mature polypeptide is longer than for other BRICHOS family members,containing 70 residues (residues 204–273), three-times longer than the BRI2 mature peptide(23 amino acids), for example. It is likely to be highly stable, owing to its fourconserved disulphide bridges  and itsOther mature polypeptides of the BRICHOS family form amyloid-like structures . This isOAF protein is a candidate biomarker for progression and clinical management of pulmonarytuberculosis and coronavirus disease 2019 (COVID-19)-associated pneumonia, owing to its1.3-fold expression levels increase in untreated tuberculosis patients versus healthycontrols  and, furOur analysis showed that structural comparison of AlphaFold models can reveal remotehomology relationships and details of polypeptide binding surfaces leading to a betterunderstanding of molecular mechanism. More specifically, the identification of a BRICHOSdomain in OAF will aid the design of future experiments that investigate its physiologicalfunction and potential role in the aetiology of amyloid-like diseases.vbac087_Supplementary_DataClick here for additional data file.


In this paper, a novel, nonbinary (NB) LDPC-coded probabilistic shaping (PS) scheme for a Rayleigh fading channel is proposed. For the NB LDPC-coded PS scheme in Rayleigh fading channel, the rotation angle of 16 quadrature amplitude modulation (QAM) constellations, 64QAM constellations and 256QAM constellations are optimized by the exhaustive search. The simulation results verify the information–theoretical analysis. Compared with the binary LDPC-coded PS scheme for Rayleigh fading channel, the proposed NB LDPC-coded PS scheme can improve error performance. In summary, the proposed NB LDPC-coded PS scheme for Rayleigh fading channel is reliable and thus suitable for future communication systems. High spectral efficiency (SE) is required in future communication systems, where advanced channel coding schemes and high-order modulation formats play an important role ,2,3. In on limit . In ordeπe/6≈1.3 dB towaon limit ,8, a binon limit ,8, is noon limit ,8. Thereon limit . Constelq) show better error performance for short blocks. p represents the number of symbolic quantization bits. The NB LDPC-coded PS scheme for an additive white Gaussian noise (AWGN) channel shows excellent error performance in [In general, the LDPC codes exhibit better behavior for high code rates when they are compared to turbo codes. In a 5G long code block coding scheme, the LDPC code is used for enhanced mobile broadband service data information. Compared to the binary LDPC codes, the nonbinary (NB) LDPC codes  over Galmance in ,12. In omance in . HoweverIn this paper, we propose a NB LDPC-coded PS scheme for Rayleigh fading channel and optimize the rotation angle of QAM signals by the exhaustive search. The theoretical average mutual information (AMI) analysis and simulation error performance show the reliability of the proposed scheme.The innovation of this paper is shown as follows.A NB LDPC-coded two-dimensional PS scheme for Rayleigh fading channel is proposed. Compared with the binary LDPC-coded two-dimensional PS scheme in , the proA NB LDPC-coded two-dimensional QAM PS scheme for Rayleigh fading channel based on signal space diversity is proposed. Compared to the NB LDPC-coded two-dimensional QAM PS scheme for Rayleigh fading channel without rotation, the proposed NB LDPC-coded two-dimensional QAM PS scheme for Rayleigh fading channel based on signal space diversity can obtain diversity gain.The rest of the paper is organized as follows. In k uniform data bits n amplitude symbols according to the unequal probability distribution The proposed NB LDPC-coded PS scheme for Rayleigh fading channel is shown in For 8-ASK constellations,For 16-ASK constellations,r, and the length of information symbols and parity symbols are X and the quadrature signal of X, respectively. In order to improve the reliability performance in fading channels, a component interleaver is used, which can make in-phase and quadrature signals go through different fading. Then, the N is the complex Gaussian noise with zero mean and variance The binary data bits lgorithm . After pIn the receiver, after passing through the phase equalizer, the received signal is converted intoThe demodulated bit probability isj-th binary mapping bit of c for According to , the decThe NB LDPC decoding symbols sequence M-ary QAM constellation consists of two orthogonal x denotes amplitude. v represents the non-negative factor, which can determine the system entropy. When The average mutual information (AMI) is the theoretical upper bound, which could be reliably transmitted for given constellation modulation formats. In this section, The regular Nyquist bit-interleaved coded modulation (BICM)-AMI and the PS BICM-AMI for Rayleigh fading channel are investigated. A Gray-labeled ribution , and PX performance of the end-to-end communication is considered. The NB LDPC codes over GF(16) have an average variable node degree In summary, compared with the binary LDPC-coded QAM with no rotation system, the proposed PS nonbinary LDPC-coded QAM with a rotation system can obtain shaping gain, coding gain and diversity gain. In In this paper, a NB LDPC-coded PS scheme for Rayleigh fading channel is proposed. The simulation results coincide well with the theoretical AMI analysis, which shows that the NB LDPC-coded PS scheme is suitable for Rayleigh fading channel, and the shaping gain can be obtained. In summary, the proposed NB LDPC-coded PS scheme for Rayleigh fading channel is robust and reliable, which is a potential scheme for the future communication system. In the future, we will consider the Rayleigh flat-fading channel with Doppler shift and consider the question of error protection inequality between the bits transmitted as the sign bits and those represented by the amplitude.


Deep6 is a deep learning model that classifies metatranscriptomic sequences as short as 250 nucleotides into prokaryotes, eukaryotes, or one of the four viral realms, using a reference-independent and alignment-free approach. Average accuracies range from 0.87 to 0.97, depending on sequence length. Viruses play key roles in marine ecosystems , but stuDuplodnaviria, Varidnaviria, Monodnaviria, or Riboviria). Deep6 is a multiclass convolutional neural network (CNN) model, consisting of 500 convolutions, 525 dense layers, and a default kernel size of 10. Four different default models accommodate input sequences of 250 to 499 nucleotides (nt), 500 to 999 nt, 1,000 to 1,499 nt, and >1,500 nt to optimize model performance for short metatranscriptomic sequences. Cellular training data are compiled from reference coding sequences (CDSs) for common marine eukaryote and prokaryote taxa , the average accuracy and group precision, recall, and derived F1 scores.Duplodnaviria, 0.65; Varidnaviria, 0.81; Monodnaviria, 0.87; Riboviria, 0.81. Where comparable, Deep6 matched or exceeded the performance of other tools. Deep6 is a high-throughput tool that can process about 100 thousand sequences per day on a single core, utilizing <1 GB of memory. Deep6 predicts viral sequences from metatranscriptomic data to support homology-based viral identification and discover novel viruses.The default models have training and validation AUROCs ranging from 0.98 to 1.00 and average accuracies ranging from 0.87 to 0.97 . Validathttps://github.com/janfelix/Deep6. Scripts are written in Python3 and rely on the readily available libraries Biopython, Keras, Sklearn, and TensorFlow. Environmental files to set up Conda environments and install dependencies are also provided. Contact J.F.F. for further information on training and cross-validation data sets.Deep6 source code and supporting information are available  at


Regulated cell death (RCD) is central to the development, integrity, and functionality of multicellular organisms. In the last decade, evidence has accumulated that RCD is a universal phenomenon in all life domains. Cyanobacteria are of specific interest due to their importance in aquatic and terrestrial habitats and their role as primary producers in global nutrient cycling. Current knowledge on cyanobacterial RCD is based mainly on biochemical and morphological observations, often by methods directly transferred from vertebrate research and with limited understanding of the molecular genetic basis. However, the metabolism of different cyanobacteria groups relies on photosynthesis and nitrogen fixation, whereas mitochondria are the central executioner of cell death in vertebrates. Moreover, cyanobacteria chosen as biological models in RCD studies are mainly colonial or filamentous multicellular organisms. On the other hand, unicellular cyanobacteria have regulated programs of cellular survival (RCS) such as chlorosis and post-chlorosis resuscitation. The co-existence of different genetically regulated programs in cyanobacterial populations may have been a top engine in life diversification. Development of cyanobacteria-specific methods for identification and characterization of RCD and wider use of single-cell analysis combined with intelligent image-based cell sorting and metagenomics would shed more light on the underlying molecular mechanisms and help us to address the complex colonial interactions during these events. In this review, we focus on the functional implications of RCD in cyanobacterial communities. Genetically regulated cell death (RCD) was first identified in metazoans and later found in prokaryotes and different eukaryotes ,2,3. TheMicrocystis) and/or exhibit multicellularity, with up to six distinct cell types in filamentous cyanobacteria  and [N] levels, phage infection could be activated, and cyanophage would actively utilize host cellular machinery to maintain and expand the phage population [Prochlorococcus, and Bar-Zeev et al. [Trichodesmium. In the latter study, recently fixed nitrogen was sinking into the sediments with the cell for further decomposition by heterotrophic bacteria. Distinct transcriptional states may co-exist during the active viral infection of cyanobacteria: (1) cells resistant to lysis that keep persisting viruses (“persisters”) and could potentially serve as an inoculum for next blooms; (2) metabolically active infected cells responsible for viral release followed by lysis, and (3) a population of cells dying without releasing any viral particles . Similarpulation . At the pulation . Fuchsmapulation  have prov et al.  for a nispeD gene—a homolog found in all marine cyanobacteria—is responsible for polyamine biosynthesis. Along with the psbA gene, also present in the marine phage genome [speD is thought to maintain the activity of the host photosystem II reaction center during phage infection [However, it is unclear what the primary factors that stabilize the host–phage relationship are and whether this relationship is cyclic . Light ae genome , if exprnfection ,148. It Microcystis-associated bacteria and the existence of complementary pathways between Microcystis and associated heterotrophs [Rhodospirillales, Burkholderiales, and Verrucomicrobiales dominated during the rapid decomposition phase, whereas Sphingomonadales, Rhizobiales, and Xanthomonadales dominated during the slow decomposition phase. Changes in a bacterial community are likely involved in the formation and maintenance of Microcystis colonial morphoforms [Microcystis was maintained in the lab culture, colonies contain heterotrophic bacteria [Sphingomonas are associated with Microcystis blooms and actively break down toxins [Microcystis colonies harbor bacterial assemblages that may be conserved across geographically distinct blooms and over different stages of blooms [Microcystis colonies harbor a selected pool of bacteria with a conserved functional potential that may help bloom persistence even under unfavorable environmental conditions. Parasite strategies, such as bacterial regulation of host RCD, can drive the evolution of host–parasite interactions leading to a possible outcome of cell death, dormancy, or infection. If bacteria delay and/or promote Microcystis colony growth and participate in colony decay, they may participate more actively in inducing cell death in the old colonies.Bloom-forming cyanobacteria are closely associated with heterotrophic bacteria ,153,154.rotrophs . The hetrotrophs  observedphoforms . These fphoforms ,158,159.bacteria . Furthern toxins ,162. Sevn toxins . Recent n toxins  confirmef blooms . This fiAnabaena, Aphanizomenon, Cylindrospermopsis, other filamentous taxa, and Microcystis [Asterionella formosa), RCD accelerates the death of infected host cells and the chytrid parasite before it completes its life cycle [In addition to well-studied viruses, many cyanobacteria species are affected by fungal parasites mainly belonging to Chytridiomycota (chytrids) . These mrocystis ,125,167.rocystis . In diatfe cycle . A fungafe cycle  and, thefe cycle  and facife cycle . HoweverEarly examination of phytoplankton automortality characterized by a loss of membrane integrity and degradation of photosynthetic activity, led scientists to supposition of a genetically based process of RCD as a source of phytoplankton mortality . CurrentInitially, the hallmarks of RCD in cyanobacteria were meant to be visualized using a standard for mammalian apoptosis reagents and techniques, such as labeling cell membrane with fluorochrome-conjugated annexin V to verify exposure of phosphatidylserine (PS) ,72,173, . During The cellular ultrastructure of cyanobacteria undergoes significant changes under RCD, and due to the bacterial size, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the major technologies of choice. A number of studies describing the detailed ultrastructure changes during cell death in cyanobacteria are available ,50,72,73The discovery of RCD-like events in prokaryotes reveals the possibility that widely divergent organisms possess conserved and previously unrecognized RCD pathways . Caspase2O2, salinity changes, and allelopathic stimuli was quantified using a microplate reader or conventional flow cytometer [N-glycanases, inducing autophagy [Most published caspase activity assays in cyanobacteria rely on commercial fluorogenic substrates that produce fluorescence signals upon proteolytic cleavage, indicating the presence of an active caspase . Fluoresytometer ,54,55,73ytometer . Howeverytometer . The panutophagy ,182,183.Recently, the first attempt to compare meta-caspase activity using specific substrates for cyanobacterial caspase-like enzymes and standard substrates for mammalian caspase evaluation was made . The desTargeting intracellular components for degradation is a crucial intracellular process that can counteract the effects of external stress such as invading pathogens . The bioWhen we had already submitted our manuscript, we came upon the interesting recent publication of Li and co-authors 2022) proposing to subdivide RCD cell types in cyanobacteria based on the involvement of caspase homologs . In our 2 proposiSynechococcus and Synechocystis spp.). The key question to be addressed is—are the data obtained with comparative genomics and supporting execution of regulated cell death in multicellular cyanobacteria applicable to unicellular cyanobacteria? We suggest that the enzymes in mechanisms in unicellular cyanobacteria are very different from those in other cyanobacteria groups. In fact, the execution of regulated cellular survival and not a regulated cellular death can be a desired outcome for these bacteria. RCD in cyanobacteria contributes to increased population survival and is an essential part of the ecological process. The multicellularity for many cyanobacterial clades suggests cell-death-related mechanisms of differentiation and protection against infection from viruses, fungi, and other pathogens. In published research, cyanobacteria species used in RCD studies are mainly represented by multicellular filamentous organisms or colonial and unicellular species having reversed from multicellularity in their evolutionary history. On the other side, there are research studies of regulated cellular survival programs in cyanobacteria, mainly using unicellular species (The existing techniques are borrowed from vertebrate RCD research, particularly from mammalian cells equipped with mitochondria—central organelles involved in their process of cell death—and are limited in their capabilities. Further RCD research in cyanobacteria requires the development of proper methods for assessment and wider use of molecular genetics and single-cell analysis in combination with cell sorting based on advanced imaging. This may lead to the discovery of novel RCD molecular pathways and regulatory and inhibitory molecules and contribute to understanding and managing bloom-forming cyanobacteria.


Here, we showthat BRICHOS, a natural anti-amyloid chaperone domain, from Bri2 efficientlyinhibits aggregation of Aβarc by mainly interferingwith secondary nucleation. This is qualitatively different from themicroscopic inhibition mechanism for the wild-type Aβ, againstwhich Bri2 BRICHOS has a major effect on both secondary nucleationand fibril end elongation. The monomeric Aβ42arc peptideaggregates into amyloid fibrils significantly faster than wild-typeAβ (Aβ42wt), as monitored by thioflavin T (ThT)binding, but the final ThT intensity was strikingly lower for Aβ42arc compared to Aβ42wt fibrils. The Aβ42arc peptide formed large aggregates, single-filament fibrils,and multiple-filament fibrils without obvious twists, while Aβ42wt fibrils displayed a polymorphic pattern with typical twistedfibril architecture. Recombinant human Bri2 BRICHOS binds to the Aβ42arc fibril surface and interferes with the macroscopic fibrilarrangement by promoting single-filament fibril formation. This studyprovides mechanistic insights on how BRICHOS efficiently affects theaggressive Aβ42arc aggregation, resulting in bothdelayed fibril formation kinetics and altered fibril structure.Amyloid-β peptide (Aβ) aggregation is oneof the hallmarksof Alzheimer’s disease (AD). Mutations in Aβ are associatedwith early onset familial AD, and the Arctic mutant E22G (Aβ Aβ42 monomers also can attach to thefibril surface and subsequently form a new nucleus (secondary nucleation)that further elongates to a fibril. The monomer-dependent fibril surfacecatalyzed secondary nucleation pathway is the main source of toxicAβ42 species.10 The Aβ42arc peptide follows a similar fibrillization mechanism as Aβ42wt, but the surface-catalyzed secondary nucleation processneeds to be treated as a multistep process as the secondary nucleationis saturated.7 Aβ42arc forms amyloid fibrils with a much faster rate compared to Aβ42wt; however, in vitro mature fibrils from both variants, fromhundreds of nanometers to a few micrometers long and 5 to 10 nm thick,share similar morphology with a twisted structure, and can form largefibril bundles.7 Recently, cryo-electronmicroscopy (cryo-EM) structure of Aβ amyloid fibrils from ADbrain tissue showed fibrils that are polymorphic with three abundantmorphologies.11 Interestingly, differenttypes of fibril arrangements have been observed from the brain ofindividuals with sporadic and familial AD, respectively.12 In vitro, for generating homogeneous Aβ42fibrils, several generations of seeding are normally applied,14 and the Aβ42 fibrils were shown to be composed of two moleculesper fibril layer, where residues 1–14 are only partially orderedand residues 15–42 form a cross-β-sheet entity with hydrophobicside chains maximally buried.14 Withoutseeding, highly homogeneous Aβ42 fibrils were formed, whichare unbranched, micrometer-long, and most of the fibrils showed arather uniform diameter of about 7 nm.15The wild-type Aβ42 BRICHOS domain fromfamilial dementia-associated Bri2 protein is efficient in inhibitingboth Aβ42wt amyloid fibril formation and neurotoxicity.23 How the BRICHOS domain interferes with familial Aβmutants with more aggressive amyloid-forming propensity, like thearctic Aβ42 mutant (Aβ42arc), remains to beelucidated.Molecular chaperonescan prevent proteins from aggregating andexerting cytotoxic effects,arc with high quality and yield and show theinhibition effectof rh Bri2 BRICHOS on Aβ42arc fibrillization kineticsand its modulation effect on the fibril morphology. The results furtherelucidate the aggregation properties of Aβ42arc andsupply a basic understanding for the effects of BRICHOS on Aβ42arc fibril formation.Here, we report a protocol for the recombinant preparationof Aβ42arc. The N-terminal globular domain (NT) of major ampullatespider silk protein (MaSp) was genetically modified, referred to asNT*Masp, and implemented as a solubility tag for producingdifferent problematic proteins and peptides.30 In the recent protocol, we applied NT* derived from flagelliformspider silk protein (FlSp), NT*FlSp, which is more solublethan NT*Masp, to generate recombinant Aβ42wt.31 Here, we follow a modified protocolwithout using urea, which might induce potential modifications tothe final product.32 Recombinant NT*FlSp-Aβ42wt and NT*FlSp-Aβ42arc were expressed in Escherichia coli, and the Ni-NTA column purified fusion proteins were subsequentlycleaved by Tobacco etch virus (Tev) protease to release the tag-freeAβ42wt and Aβ42arc peptides withoutany extra amino acid residues (S1a). The Aβ42wt and Aβ42arc monomers were isolated viasize exclusion chromatography (SEC), which showed good quality interms of purity (S1b). To obtain pure Aβ42arc monomers for kinetic analysis, the SEC-isolated [superdex30column (26/600)] monomers were lyophilized, solubilized with guanidiumchloride, and isolated again by SEC using a superdex30 column (10/300),which showed very well-separated monomer and oligomer peaks . The final yield of NT*FlSp-Tev reached 145mg per liter LB medium and showed high purity . The NT*FlSp-Tev fusion protein presentedvery good cleavage efficiency against NT*FlSp-Aβ42wt. The cleavage reaction was performed in the cold room atan enzyme to a substrate ratio of 1:100 (w/w) where the half-timefor cleavage was estimated to be ∼3.2–4.1 h . No visible protein aggregationwas seen, and no aberrant degradation appeared as judged by SDS-PAGE, indicating that fusion toNT*FlSp tag can enhance the stability of Tev and does notimpair Tev activity.First, we set out to establishan efficient and robust protocol for recombinant production of Aβ42er peaks 1d, indicarc and Aβ42wt, we used thioflavin T (ThT)33 to monitor the fibrillization kinetics as afunction of time at a range of different initial monomer concentrations.Both Aβ42arc and Aβ42wt showed typicalsigmoidal aggregation kinetics (S3a), andthe fibrillization half-time, τ1/2, increased withdecreasing monomer concentrations, while the maximum rate of aggregation, rmax, decreased for each of the kinetics models and can be used to indicate the dominantmechanism of aggregation.34 The aggregationhalf-time and the initial monomer concentration were plotted on adouble logarithmic scale, and Aβ42arc showed a γvalue of −0.8 ± 0.1, while for Aβ42wt, it was −1.4 ± 0.1  and secondary nucleation  as well as elongation (k+),34 and the combined rateconstants 37 Global fitting with combined rate constants wt aggregationtraces could be sufficiently described by secondary nucleation dominatedmodels , whereas Aβ42arc traces were fitted with an additional Michaelis constant S3c), indicatingthat saturation of secondary nucleation applies to Aβ42arc fibrillization. The global combined rate constants arc aggregationtraces were 2.3 and 6.0 times higher, respectively, than that forAβ42wt, indicating that the Arctic mutation acceleratesAβ42 peptide aggregation through predominantly secondary pathways.To further investigate the relationship between the initial monomerconcentration and the final fluorescence intensity, the final intensitieswere plotted as a function of the initial monomer concentrations,which exhibited a linear relationship for both Aβ42arc and Aβ42wt    3. Under ilaments 3a–c. Theilaments 3a–c, rep curlier 3d,e. Intpresent 3d,e.We present 3h,indicng axis) 3f,g that peptide3a–c.Thi fibrils 2d.wt peptide,39 but it is not evident whether BRICHOS hasthe ability to suppress also Aβ42arc aggregationsince its aggregation mechanism is considerably different from Aβ42wt. To evaluate the inhibition effects of rh Bri2 BRICHOS onthe fibrillization process of Aβ42arc, monomericrh Bri2 BRICHOS species were isolated by SEC and added to Aβ42arc. In line with previous studies,39 rh Bri2 BRICHOS showed efficient inhibitionof Aβ42wt fibrillar aggregation, as indicated bylinearly increased τ1/2 and mono-exponentially declined rmax with increased BRICHOS concentrations . Although Aβ42arc showed substantially faster aggregation than Aβ42wt , indicating that rh Bri2 BRICHOSmonomer mainly interferes with the secondary pathway rather than theprimary pathway of Aβ42wt fibril formation, as proposedpreviously.21 A similar mechanism but withan additional secondary nucleation saturation effect (a multistepdominated secondary nucleation model) was applied for Aβ42arc in the presence of rh Bri2 BRICHOS monomers. Also for Aβ42arc, a noticeable decrease in Figure S3g,h). These results indicate that rhBri2 BRICHOS possesses the capacity to suppress Aβ42arc assembly into fibrils, by mainly interfering with the secondarypathway.The Rh Bri2 BRICHOS domain has been shown to inhibit amyloid fibrilformation of several peptides efficiently, including Aβ42trations S3d,e. Altrations S3d,e. Altrations S3d,e. Alarc fibril formation, wecarried out aggregation kinetics with a high seed concentration. Aggregationtraces typically display a concave aggregation behavior under suchconditions of Aβ42arc peptide is primarily blocked by rh Bri2BRICHOS, and only a small effect is visible on the elongation rate k+.To figure out which of the microscopic events areaffected by rhBri2 BRICHOS against Aβ42nditions 4d, where Aβ42arc 4e, whicharc fibrils (k2) is inhibited, but it is increased when elongation(k+) is blocked.41 It has been shown that rh Bri2 BRICHOS monomers can reduce nucleationunit generation by 70% during Aβ42wt fibril formation,23 while the rh proSP-C BRICHOS, mainly blockingthe secondary nucleation of Aβ42wt fibrillization,exhibits an efficiency of 80%.41 To illustratethe generation of nucleation units during Aβ42arc fibrillization in the presence or absence of rh Bri2 BRICHOS monomers] resultedin the fact that more single-like (S) fibrils were observed  with BRICHOS were narrower compared to the Aβ42arc alone fibrils (arc fibrils [(Aβ42arc)fibril + BRICHOS].Under TEM  intertwined filaments with twists were observed ,48 while proSP-C BRICHOSspecifically inhibits secondary nucleation.41 Recently, Bri2 BRICHOS has been shown to affect both Aβ42wt secondary nucleation and elongation;21 however, this situation is changed for Aβ42arc, where mainly secondary nucleation but not the elongation was affected, and cleaved by thrombin, and eventuallythe tag-free Bri2 BRICHOS monomers were isolated by a Superdex75 column(Cytiva), as described in previous study.21 The 42 amino acid residues (1–42) of Aβ were fusedto the NT*FlSp tag and expressed in BL21(DE3) E. coli.31 In brief, theNT*FlSp-Aβ42wt was purified with a Ni-NTAcolumn with following the protocol, as described previously31 but without using denaturant  toavoid potential urea-induced modification. The fusion NT*FlSp-Aβ42wt proteins were cleaved by NT*FlSp-Tev and lyophilized. The lyophilized powder was solubilized in 20mM Tris pH 8.0 with 7 M guanidium chloride, and the Aβ42wt monomers were isolated by a Superdex30 26/600 (Cytiva) in20 mM NaPi pH 8.0 with 0.2 mM EDTA and aliquoted in low-binding Eppendorftubes (Axygene). The Aβ42wt concentration was calculatedthrough an extinction coefficient of 1424 M–1 cm–1 for (A280–A300). For generating arctic mutant(E22G) of Aβ42, the primers 5′-ctggtgttcttcgctggagacgtgggttctaac-3′and 5′-gttagaacccacgtctccagcgaagaacaccag-3′ were synthesized.With the NT*FlSp-Aβ42wt plasmid as thepolymerase chain reaction (PCR) template, NT*FlSp-Aβ42arc was obtained with the QuikChange II XL site-directed mutagenesiskit . The preparation of Aβ42arc monomerswas performed with following the same protocol as described above,but the final Aβ42arc monomers were refined withan analytical superdex30 10/300 column (Cytiva). Regarding the Tevconstruct, gene coding for Tev proteinase was cloned into the modifiedpET vector with NT*FlSp solubility tag, encoding the fusionprotein NT*FlSp-Tev. NT*FlSp-Tev plasmid wastransformed into BL21(DE3) E. coli competentcells, which were cultured at 37 °C in LB medium with 70 μg/mLkanamycin until an OD600nm ∼ 0.8. The temperaturewas turned down to 20 °C, and 0.5 mM  isopropylβ-d-1-thiogalactopyranoside was added for overnightexpression. The cells were collected by 7000 g centrifugationat 22 °C for 20 min and resuspended in 50 mM NaPi pH 8.0 with200 mM NaCl and 10% glycerol. After 5 min on ice sonication , the cell lysate was centrifuged for 30 min at 4°C with a speed of 24 000 g, and NT*FlSp-Tev present in the supernatant was purified with a Ni-NTA column.The final target proteins were eluted by 50 mM NaPi pH 8.0 containing200 mM NaCl, 10% glycerol, and 250 mM imidazole and immediately buffer-exchangedto 25 mM NaPi pH 7.5 with 100 mM NaCl and 10% glycerol with a HiPrep26/10 desalting column (Cytiva). The cleavage efficiency was evaluatedby cleaving NT*FlSp-Aβ42wt at a ratioof 1:100  at 4 °C via analyzing bandintensities at different time points on SDS-PAGE. For all the constructsabove, the final DNA sequences were confirmed by sequencing .The recombinantprotein NT*wt  and Aβ42arc  at differentconcentrations in the presence of 10 μM ThT were added to eachwell of half-area 384-well black polystyrene microplates with clearbottom and nonbinding surface  and incubatedat 37 °C under quiescent conditions. The ThT fluorescence wascontinuously recorded using a 440 nm excitation filter and a 480 nmemission filter . For investigatingthe inhibition effects of rh Bri2 BRICHOS monomers on Aβ42arc fibril formation, 20 μL of solution (20 mM NaPi pH8.0 with 0.2 mM EDTA) containing Aβ42arc monomers,10 μM ThT, and different concentrations of rh Bri2 BRICHOS monomersat molar ratios 0, 10, 50, 70, and 100% relative to the Aβ42arc monomer concentration were added to each well of half-area384-well black polystyrene microplates with clear bottom and nonbindingsurface  and incubated under quiescent conditionsat 37 °C. The fluorescence was recorded as described above. Toprepare fibrils for EM observation of both Aβ42wt and Aβ42arc fibrils, 20 μL of solution (20mM NaPi pH 8.0 with 0.2 mM EDTA) containing 3.0 μM Aβ42wt or 3.0 μM Aβ42arc monomers with andwithout 100% BRICHOS was added to each well (four replicates) of half-area384-well black polystyrene microplates with clear bottom and nonbindingsurface  and incubated at 37 °C underquiescent conditions overnight, among them one well for each was addedwith 10 μM ThT to monitor the aggregation. Furthermore, 100%(molar ratio) of rh Bri2 RRICHOS monomers were added to each wellafter the formation of fibrils and incubated again at 37 °C underquiescent conditions overnight. For investigating Aβ42 fibrillizationkinetics with seeds, 20 μL of solution containing 10 μMThT, 3 μM Aβ42 monomer, different concentrations of monomericrh Bri2 BRICHOS, and 0.6 μM seeds  were added in cold room to each wellof half-area 96-well plates and incubated at 37 °C under quiescentconditions. The fluorescence measurement settings were carried outas described above. Linear fits were applied to the concave aggregationtraces (the first 24 min) to determine the initial slopes. For allthe experiments, aggregation traces were normalized and averaged usingfour replicates.For monitoring amyloid fibril formation andthe kinetics, 20 μL of solution (20 mM NaPi pH 8.0 with 0.2mM EDTA) containing monomeric Aβ42arc) with BRICHOS addedinitially and after fibril preformed, respectively, were applied toform var-coated nickel grids and incubated for 2 min. Excess solutionwas removed with the filter paper . Blocking wasperformed by incubating the grids for 30 min in 1% BSA in TBS , followed by 3 × 10 min TBS washing. The grids were thenincubated with primary antibody  in cold room overnight, followed again by 3 ×10 min TBS washing. The grids were incubated with 10 nm gold particle-coupled secondary antibody at roomtemperature for 2 h and then washed with 1× TBS for 5 ×10 min. For staining, 2.5% uranyl acetate (2 μL) was added toeach grid (for 20 s), and excess solution was carefully removed. Thegrids were air-dried and analyzed by TEM (Jeol JEM2100F at 200 kV).For imaging fibrils of Aβ42wt and Aβ42arc co-incubated with and without rh Bri2 BRICHOS monomersor with added BRICHOS to the preformed fibrils, the final incubationproducts were applied to carbon-coated copper grids  and incubated for 2 min. Excess solution was removed byblotting with the filter paper , and the grids werewashed with two drops of Milli-Q water. For staining, 7 μL of2% uranyl acetate was added to each grid for 45 s before final blottingand air-drying. The grids were analyzed by TEM (Jeol JEM2100F at 200kV). All measurements were performed using ImageJ 1.53k. The single-likefibers with no visible twists or bundle structures were classifiedas S, whereas the multiple fibrils were classified as M. The measurementsof twist body and twist body (crossover point) of Aβ42wt fibrils included 39 and 40 points, respectively. For Aβ42arc fibrils, 223 and 65 measurement points, respectively, wereselected randomly for the diameter measurements. For Aβ42arc and rh Bri2 BRICHOS co-incubated fibrils, the diametermeasurements of single-like and multiple fibrils were performed on77 and 76 measurements, respectively.For immunogold staining of Aβ42 fibrils, the final incubationproducts , is describedby an integrated rate law, as described by Cohen et al.53kn, k+, and k2 are the microscopic rate constants for primary nucleation,elongation, and secondary nucleation, respectively, and nC and n2 arethe reaction orders of primary and secondary nucleation, respectively.The aggregations trace of Aβ42wt and Aβ42arc with and without rh Bri2 BRICHOS monomers were globallyfitted using IgorPro and the AmyloFit 2.0 platform34 (https://amylofit.com/amylofitmain/fitter/) with models forsecondary nucleation dominated (unseeded) and multistep secondarynucleation dominated (unseeded) according to the γ values andprevious reports,7 respectively, wherethe k+kn and k+k2 were constrained globally or free for aggregation traces with BRICHOS.The parameters nC and n2 both were set to 2. The nucleation unit generationwas calculated by integrating the nucleation rate rn(t) over the reaction,41 where rn(t) = knm(t)nC + k2M(t)m(t)n2.For extracting the aggregation half-timeτt test(unpaired) was used for statistical analysis of two groups of data.The multiple groups were statistically compared with the ordinaryone-way analysis of variance following by multiple comparisons withTukey correction. Significance levels are *p <0.05; **p < 0.01; ***p < 0.001;and ****p < 0.0001.All the statistically analyseswere performed in Prism 9. Student’s All data and materials related tothis paper are available uponrequest.