Boophone disticha, Leonotis leonurus and Mentha longifolia were identified as the three most popular plants, with over eight mentions each from the ethnobotanical records. The dominant families were Asteraceae (24), Fabaceae (16), Amaryllidaceae (10), and Apocynaceae (10) which accounted for about 32% of the 186 plants. Only 27 (≈14.5%) of the plants have been screened for antidepressant activity using in vitro and in vivo models. Agapanthus campanulatus, Boophone disticha, Hypericum perforatum, Mondia whitei and Xysmalobium undulatum, represent the most studied plants. Phytochemical investigation on nine out of the 27 plants revealed 24 compounds with antidepressant-like effects. Some of these included buphanidrine and buphanamine which were isolated from the leaves of Boophone disticha, Δ9-tetrahydrocannabinol, cannabidiol and cannabichromene obtained from the buds of Cannabis sativa and carnosic acid, rosmarinic acid and salvigenin from Rosmarinus officinalis, A significant portion (≈85%) of 186 plants with ethnobotanical records still require pharmacological studies to assess their potential antidepressant-like effects. This review remains a valuable reference material that may guide future ethnobotanical surveys to ensure their robustness and validity as well as database to identify promising plants to screen for pharmacology efficacy.Globally, the search for safe and potent natural-based treatment for depression is receiving renewed interest given the numerous side-effects associated with many existing drugs. In South Africa, the use of plants to manage depression and related symptoms is fairly documented among different ethnic groups. In the current study, we reviewed existing ethnobotanical, ethnopharmacological and phytochemical studies on South African medicinal plants used to manage depression. Electronic databases were accessed for scientific literature that meets the inclusion criteria. Plants with ethnobotanical evidence were subjected to a further pharmacological review to establish the extent (if any) of their effectiveness as antidepressants. Critical assessment resulted in 20 eligible ethnobotanical records, which generated an inventory of 186 plants from 63 plant families. Due to the cultural differences observed in the definition of depression, or lack of definition in some cultures, most plants are reported to treat a wide range of atypical symptoms related to depression.  Clinical depression represents one of the most prevalent and highly comorbid psychiatric conditions that causes a negative emotional experience associated with pathophysiological changes . In thisMDD is one of the three leading causes of disease burden in South Africa and globally . The WHOMDD-com includes mental health related problems that could be classified as depression based on similar clinical symptoms and is perceived by African traditional healers as caused by ancestors or bewitchment . When inBoophone disticha is one of the most popular medicinal plants in South Africa where bulb infusions of this plant are used to treat headache and mental condition  N.E. Br has been developed into various pharmaceutical products for mood elevation  screening and phytochemical assessment. This study is envisaged to provide an in-depth and current state of knowledge in the search for South African plants with antidepressant potential and identify critical gaps for future research direction in the on-going global search for safe and efficient antidepressants.Despite the anecdotal and long-term use of medicinal plants for the treatment of MDD and related ailments in South Africa, an in-depth and up-to-date appraisal on this subject is lacking. Moreover, the need to bridge the cross-cultural gap observed in the definition of depression by exploring the regional, cultural and/or traditional concepts of depression as understood by South African traditional healers cannot be overemphasized. This review focuses on the ethnobotanical studies on South African plants with potential antidepressant effects, their pharmacological  in South Africa• Be published or made available on the internet during the research period For a research article to be included in the review it must:in vitro.• Plant extracts must be investigated against either Serotonin reuptake transporter (SERT), Dopamine transporter (DAT) and Noradrenalin transporter (NAT) receptors • Evaluate the effect of plant extract on behavioral markers of depression in vivo and the safety/toxicity profile.For Ethno-Pharmacological Studies• Ethnobotanical review articles (literature or systematic)• Focus on natural resources (other than plants) used for depression• Ethnobotanical surveys that is not focusing on South Africa• Have limited data (e.g. missing scientific plant names) on the medicinal use of plants for depressionResearch articles were excluded from the review if they:http://theworlflora.online) and PlantZAfrica (http://pza.sanbi.org/) while the local names were confirmed using PlantZAfrica (http://pza.sanbi.org/). Any botanical synonyms or unaccepted names were updated to the recent updated plant nomenclature and are reported as such in this review. The plants listed in this review are arranged in alphabetical order based on plant families and scientific names.Relevant data on the antidepressant-like effects of South African medicinal plants were extracted to Excel spreadsheets following the pre-defined criteria. Bibliographies from accessed articles, together with their citations, were downloaded and saved on an online reference manager (EndNote). Missing information from some articles  and in cases of research papers lacking geographic locations of the study, the data was retrieved through direct web searching (Google). Scientific names of plants, family, local names, life form of plants, method of preparation of the plant-based medicine, route of administration and short notes were collected for each plant from each article. Where possible, data on the ethnic group that uses the plants traditionally were collected. Plant species and family were validated in references to The World Flora Online  retrieved from the NWU library, making a total of 1,448 records that were screened. After the removal of duplicates, 44 articles and books were assessed for eligibility and explored relative to the inclusion criteria. The full text of 44 studies was reviewed in detail and 24 studies were removed due to limited data and other reasons stated in the exclusion criteria. Finally, 20 studies were reviewed for the documentation of the potential antidepressant-like effects of medicinal plants . The litThe operational definition of MDD was inconsistent across most of the ethnobotanical literature reviewed. Most of the literature did not define MDD but a broad range of traditional medicinal uses in their surveys, rather than medicinal uses specific to MDD. Since most medicinal uses reported in the literature are indigenous knowledge gathered from South African traditional healers, records of the potential antidepressant effects of medicinal plants are listed as MDD-com, which entails ailments based largely on somatic symptoms such as headaches, fatigue, or spirits. According to The absence of methodological framework was evident in one of the reviewed study . A numbeAdenia gummifera (Harv.) Harms, Albizia adianthifolia (Schum.) W.Wight, Ballota hirsuta Benth., Boophone disticha, Gomphocarpus fruticosus (L.) W.T.Aiton, Mentha longifolia (L.) L. and Leonotis leonurus (L.) R.Br.) were categorized as the most popular based on a relatively high number of mentions in the ethnobotanical surveys reviewed. The three most popular plants, with over eight mentions each from ethnobotanical surveys, included Boophone disticha, Leonotis leonurus and Mentha longifolia. Several other plants, such as Agapanthus campanulatus, Cannabis sativa, Heteromorpha trifoliata (H.L.Wendl.) Eckl. and Zeyh., Sceletium tortuosum, Schinus molle L. and Xysmalobium undulatum, have been used traditionally in South Africa to treat MDD and MDD-com  Eckl. and Zeyh. are administered for mental and nervous diseases and smoked for headaches  being the most predominantly used plant part . HoweverA total of 63 plant families are being used in South Africa to treat MDD and MDD-com . Twenty-Senecio (Asteraceae), one of the largest genera of flowering plants distributed worldwide. Centaurea cyanus L. (Asteraceae) using preparative RP-HPLC analysis and afforded four alkaloids of the indole variety: moschamine, cis-moschamine, centcyamine and cis-centcyamine. Existing pharmacological studies have reported the antidepressant-like activity of Artemisia dracunculus L. (Asteraceae) in animal models of depression  as a plant family is known to have diverse therapeutic applications, and has a long history in traditional medicine . Memberspression . Althougunculus . The preAlbizia adianthifolia (Fabaceae) leaves increased swimming time and decreased immobility time in the forced swimming test conducted using male Wistar rats , a popular plant in South African traditional medicine, led to the identification of buphanamine, buphanisine, buphanidrine, distichamine and crinine and a confirmation of its antidepressant effects  Skeels commonly known as White’s ginger, is a popular South African plant used in folk medicine to treat diseases of the nervous system and it has demonstrated antidepressant properties under in vitro conditions -loliolide.Apocynaceae as a plant family is an important and popular source of a number of drugs including simple indoles, carbolines, steroidal amines, isomeric quinindolines and quinindoles and miscellaneous type alkaloids . Membersnditions . Koorban to SERT . Ethanolpression . The autin vitro and in vivo investigations that evaluated the potential antidepressant effects of medicinal plants. An aqueous extract from Datura ferox L. seeds exhibited high affinity the SERT binding assay (Datura stramonium L. exhibited antidepressant-like effects in the forced swimming test and open field test conducted by Sceletium tortuosum (L.) N.E. Br.), Cannabaceae (Cannabis sativa L.), Hypericaceae (Hypericum perforatum L.), Pteridaceae (Adiantum capillus-veneris), Lauraceae (Cinnamomum camphora), Poaceae (Cymbopogon nardus), Capparaceae (Maerua angolensis), Meliaceae (Melia azedarach), Polygalaceae (Securidaca longipedunculata), Rhamnaceae (Ziziphus mucronata) and Anarcadiaceae (Schinus molle)  .To allow for easy identification and selection of potential medicinal plants for pharmacological investigation, the relationship between plant families with potential antidepressant effects and the various phytochemicals present in plants belonging to these families remain pertinent. This was achieved by looking closely at the phytochemical profiles of medicinal plants belonging to the four plant families with the highest number of plants recorded in this review. Based on the fact that plants in the same family may have similar phytochemical profiles, it is hypothesized that recognizing plant families of potential  could lead to the discovery of potential medicinal plants within those families for pharmacological investigations using models of MDD. This approach may provide insight on the potential presence of novel phytochemical compounds of antidepressant value not previously isolated from medicinal plants and stimulate alternative antidepressant drug discovery.. These plants were represented by 12 pharmacological studies previously conducted to investigate the in vitro and in vivo antidepressant-like effects. Medicinal plants with the highest number of phytochemicals identified are Boophone disticha and Rosmarinus officinalis, each with five phytochemicals isolated, identified and their antidepressant effects investigated in various models of depression  re-uptake activity , cannabidiol (CBD) and cannabichromene (CBC) from the buds of Cannabis sativa, the three phytochemicals that showed antidepressant-like effects in two animal models of depression (the force swimming test and the tail suspension test) conducted in the same study. Other phytochemicals isolated from indigenous plant extracts in similar studies, such as (R)-Citronellal, asiaticoside, adhyperforin, hypericin, loliolide and carnosic acid, have been investigated in various models of depression, where they showed positive antidepressant-like results , while the most popular method of extraction was maceration, which was used in four of the studies (33.3%). Various methods were used to isolate and identify phytochemicals from plant extracts, and these include HPLC–UV separation, bioassay-guided fractionation, vacuum liquid chromatography, gas chromatography, column chromatography and preparative thin layer chromatography. Using bioassay-guided fractionation on VLC and preparative TLC,  results . The use results .Agapanthus campanulatus, Haemathus coccineus, Scadoxus puniceus, and Mondia whitei have been confirmed to possess antidepressant effects, however, they are documented ethnobotanically to be administered in the treatment of mental disorders, insanity, headache, nervous disorder, used as tranquilizers, snuff and sedatives , noradrenergic (NAT) and dopaminergic (DAT) receptors  protein binding assay, the functional SERT uptake inhibition assay, the functional DAT (dopamine transporter) uptake inhibition assay and the functional NAT  uptake inhibition assay. The SERT binding assay includes, briefly, mixing a dilution of the extract with [3H] citalopram and rat brain tissue suspension, replacing the extract with paroxetine for positive control and with a buffer for negative control. All samples are then incubated for 2 h and filtered under vacuum before radioactivity is measured by liquid scintillation , tail suspension test (TST) and the open-field test (OFT) were applied on in vivo models the investigation of potential antidepressant effects of medicinal plants. The forced swimming test is the most widely used test for screening of antidepressants and it involves placing rats or mice individually in plastic cylinders containing a column of water with no possible escape for 6 min   . A studyand TST) . This senditions . Oleurop and OFT .Agapanthus campanulatus, Boophone disticha, Hypericum perforatum, Mondia whitei and Xysmalobium undulatum. These plants were investigated in all the bioassays included in this study. Based on the plant parts screened, leaf extracts for 11 plants  demonstrated antidepressant activity. These studies serve as scientific rationale for the utilization of these pharmacological assays. Furthermore, we provides insight on the plant parts and solvents used in the preparation of anti-depressive plant extracts for pharmacological investigation.The most extensively studied plants included Boophone disticha, Haemanthus coccineus and Scadoxus puniceus have the isoquinoline alkaloids as the toxic compound characterized by symptoms such as dizziness  have been investigated for their antidepressant-like activity in


Cerebral microvascular rarefaction, the reduction in number of functional orstructural small blood vessels in the brain, is thought to play an importantrole in the early stages of microvascular related brain disorders. A betterunderstanding of its underlying pathophysiological mechanisms, and methods tomeasure microvascular density in the human brain are needed to developbiomarkers for early diagnosis and to identify targets for disease modifyingtreatments. Therefore, we provide an overview of the assumed mainpathophysiological processes underlying cerebral microvascular rarefaction andthe evidence for rarefaction in several microvascular related brain disorders. Anumber of advanced physiological MRI techniques can be used to measure thepathological alterations associated with microvascular rarefaction. Althoughmore research is needed to explore and validate these MRI techniques inmicrovascular rarefaction in brain disorders, they provide a set of promisingfuture tools to assess various features relevant for rarefaction, such ascerebral blood flow and volume, vessel density and radius and blood-brainbarrier leakage. The human cerebral microcirculation consists of approximately 600 kilometersof capillaries, and the number of endothelial cells in the brain is very similar tothe number of neurons. Since the brain has little reserve capacity, a reduction in perfusion isdeleterious. There is growing evidence that alterations at the level of the cerebralmicrocirculation play a key role in vascular brain diseases andneurodegeneration.4Normal brain function requires a very high energy supply, as is illustrated by thefact that the brain uses about 20% of total body energy even though it comprisesonly 2% of total body mass. Microvessel density decreases with normal aging, but this process isaccelerated in microvascular brain disorders. In pathology studies on post-mortem specimens, cerebral microvascularrarefaction has been demonstrated in animal models of ageing, Alzheimer’s disease(AD), obesity, hypertension and diabetes mellitus, and the same has been found inaging humans, AD patients, and humans with white matter hyperintensities (WMH).Importantly, small vessels such as arterioles and capillaries undergo a reduction intheir number, a phenomenon called rarefaction .5 Micr A reduction in perfusion likely precedes structural rarefaction. Reduced perfusion may eventually lead to neural dysfunction andmicrostructural tissue damage, which contribute to neurodegeneration and macrostructural lesions visible onmagnetic resonance imaging (MRI).Microvascular rarefaction may reveal itself as a functional or structural phenomenon.Functional rarefaction reflects a reversible reduction in perfused microvessels,whereas structural rarefaction reflects an actual anatomical loss of microvessels.Investigating microvascular density directly in the human brain is challenging, sincepathological examination is only possible post-mortem, and the spatial resolution ofthe existing medical imaging machines is insufficient for direct visualization ofcerebral capillaries as these are only about 10 µm in diameter. Nonetheless, sincestructural rarefaction is preceded or paralleled by functional rarefaction andmicrovascular dysfunction, this opens up avenues for measuring rarefaction byassessing microvascular perfusion and function.In this narrative review, we provide an overview of the literature on microvascularrarefaction in the human brain. A better understanding of the relevance ofmicrovascular rarefaction, its underlying mechanisms, and methods to measuremicrovascular density in the brain, is needed to develop markers for early diagnosisand to identify targets for disease modifying treatments. We will first discusspathophysiologic mechanisms underlying cerebral microvascular rarefaction, andevidence of microvascular rarefaction in several brain disorders. Thereafter,relevant principles of advanced MRI techniques that can be used to study rarefactionin the human brain including their limitations are explained. We have limited thescope of this review to MRI approaches that probe physiologic properties in humanvascular disease and aging, since MRI is the most promising and versatile imagingtechnique to measure rarefaction, as it can be repeated over time to monitorchanges, and can provide a variety of functional measures on themicrocirculation. which is a constellation of microstructural elements that regulates the closerelationship between metabolic demand, blood supply and neuronal activity in thebrain  andinsulin like growth factor-1 (IGF-1), are essential for endothelial cellsurvival and proper microvascular function, and are known to be dysregulated inmicrovascular rarefaction. This implies defects in angiogenesis cannot account for the initiation ofcapillary rarefaction, though they can account for failed repair response aftermicrovascular rarefaction has occurred and a state of hypoxia is present. Activecapillary regression is now increasingly being recognized as important inmicrovascular rarefaction though its molecular mechanisms remain poorlyunderstood. Actively regressing vessels can be identified by string vessels,which are basement membranes without endothelial cells  showed an increased number of stringvessels in the cortex and hippocampus in comparison with wild type mice. Of note, TGF-β1 is shown to be increased in the cerebrospinal fluid andblood of AD and vascular cognitive impairment (VCI) patients.25Furthermore, post-mortem studies that compared the brains of AD patients withelderly controls have shown string vessels to be increased in AD.2630 On the contrary, stringvessels were found to be decreased in cerebral white matter in subjects with WMH. However, since it is known that string vessels gradually disappear withtime, and the observed decrease in string vessels was greater than the decreasein capillaries, it was hypothesized that an early loss of capillaries in WMH isfollowed by the disappearance of string vessels, so that by the time the subjectdies there are few string vessels left.Active regression is not simply the result of a lack of pro-angiogenic factors.Indeed, merely reducing VEGF is not sufficient to initiate or induce capillaryregression, and even in the presence of relatively high levels of VEGF, vesselregression occurs. Endothelial dysfunction has been shown to correlate with functional rarefaction, and apoptotic endothelial cell death is involved in structural capillary rarefaction. Endothelial injury causes a decreased bioavailability of nitric oxide(NO), resulting in a reduced arteriolar vasodilator capacity, leading todecreased blood flow through the capillaries, i.e. functional rarefaction. Decreased NO production also promotes endothelial apoptosis, and has been causally linked to an impaired angiogenic capacity ofendothelial cells, thereby directly contributing to structural rarefaction as well.Apoptosis of a relatively small proportion of endothelial cells may besufficient to mediate significant microvessel rarefaction. The importance of oxidative stress in the origin of endothelial injury inthe brain is supported by studies showing that attenuation of mitochondrialoxidative stress using different inhibitors of ROS production increases cerebralcapillary density, restores angiogenic potential in aged rodents,40 andrestores endothelium-mediated vasodilation in the brain of aged mice.42Cumulative exposure to cardiovascular risk factors leads to oxidative stress byreactive oxygen species (ROS), causing endothelial dysfunction and apoptosis. the loss of pericytes results in dilated capillaries, leading to unevenand disrupted flow patterns within the network, causing disruptions in capillarytransit time. Thus, pericytes may play an important role in the functional stage ofmicrovascular rarefaction. Furthermore, when a disturbance in thepericyte-endothelial cross-talk appears, pericytes detach from the vascular walland migrate into the interstitial space, leaving the vascular wall unprotectedand vulnerable. In the interstitial space, pericytes undergo a functional transition tomyofibroblasts (scar-forming cells)  or undergo apoptosis. Preliminary studies have shown that myofibroblasts may promote vascularleakage and the death of capillaries.48 Hence, pericytedetachment is deleterious to capillaries in multiple ways and the loss ofpericytes results in structural rarefaction. Pathways involved inpericyte-endothelial cell disassembly have been reviewed in detail before andwill not be discussed here further. Lastly, pericytes might serve as a local stem cell population thatreplenish differentiated interstitial and vascular cells lost during aging. The loss of this reparative capacity also promotes capillary rarefaction.Indeed, in mice models, a reduced pericyte coverage and pericyte number in thebrain have repeatedly been correlated with reduced microvasculardensity.51 A postmortem study showed a reduced microvessel densityin combination with marked degeneration of pericytes in the hippocampus andcortex in AD patients in comparison with controls.One of the functions of pericytes is to control capillary tone. Whereas enhancedactivity of pericytes causes capillary contraction and entrapment of erythrocytes, and is associated with cardiovascular risk factors such as hypertensionand diabetes.55 Microemboli are associated with both AD and vascular dementia. Decreased shear stress leads to compromised endothelial NO synthase(eNOS) function in endothelial cells resulting in lower levels of NO, whichinitially leads to prolonged periods of functional rarefaction by compromisedvasodilation, and eventually results in structural rarefaction. Additionally, mechanical forces may play a significant role inpro-angiogenic and anti-angiogenic reprogramming. Cyclic strain triggersendothelial cell programs which result in sprouting angiogenesis and recruitmentof smooth muscle cells. For example, it has been shown that a lack of blood flow results in lossof availability of VEGF. Flow dependent shear forces also provide the survival signal forendothelial cells and a decrease in shear stress in capillaries facilitatesendothelial apoptosis. As such, it has been shown that shear stress generated by a flow chamberattenuates brain microvascular endothelial cell apoptosis in rats. Hence, a loss of shear stress may present a mechanism linking functionaland structural rarefaction.Stagnation of blood flow leading to a reduction of shear stress can be caused bytortuosity of microvessels and vascular blockage by spontaneous microemboli.Tortuosity of microvessels occurs with aging  Endothelial cells are also the key cell type involved in angiogenesis,hence endothelial malfunction or death will also result in impaired angiogenesis. A decrease in shear stress, on its turn, leads to impaired angiogenesisand endothelial dysfunction and apoptosis,59 rather than directlycausing rarefaction. This interdependency leads to self-reinforcement, wheredysfunction in one cell type sets of a chain of events causing furtheracceleration of the pathophysiological process. Although a complex interactionbetween these processes is probably present, most studies have mainly focused ona singular process. It is for this reason that the exact sequence and relativeimportance of events in the origin of microvascular rarefaction in the humanbrain remains elusive.To provide a structured overview of the pathophysiological pathways involved inbrain microvascular rarefaction, we presented the four main pathophysiologicalprocesses separately. However, these processes are not self-containing, yetthere is a continuous interplay between these processes. Pericytes, for example,are dependent on cross-talk with endothelial cells for proper functioning, soendothelial dysfunction or apoptosis will typically be accompanied by pericyte loss.2652 On the other hand, forimpaired angiogenesis, endothelial dysfunction and loss of shear stress,‘intervention’ studies in animals have been performed to intervene in thepathophysiological process resulting in higher microvascular density,1240 and these could beregarded as a high level of evidence for the role of these processes.Furthermore, most evidence for these pathophysiological processes ofmicrovascular rarefaction in the brain comes from animal studies, which can beregarded as low level evidence, since it is unclear how findings in animalsrelate to findings in humans. Although far more research has been conducted inimpaired angiogenesis than in pericyte loss in animal models, pericyte loss andvessel regression have been confirmed in human post-mortem case-controlstudies. One of the typical structural brain lesions that can be seen on MRI are WMH. The precise pathways in microvascular dysfunction leading to cSVD remainunclear, but there is growing evidence that microvascular rarefaction plays acontributing role. Postmortem studies showed that subjects with WMH had adecreased cerebral microvascular density in comparison with subjects withoutWMH.63 In a mouse model of cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), amono-genetic type of cSVD, a substantial reduction in white matter capillarydensity was shown, and this rarefaction was shown to precede the occurrence ofWMH and to be progressive. A decline in cerebral microvascular density has also been correlated withdiminished cognitive function in mice.cSVD is an umbrella term that covers all pathologies of the cerebral smallvessels. It causes hemorrhagic strokes, ischemic lacunar strokes, VCI and dementia.6570 Since these comorbiditiesare strongly linked to the development of age- and vascular risk factorassociated cSVD and have been shown to cause cerebral microvascular rarefaction,this also supports a role of rarefaction in the development of cSVD.Additionally, microvascular rarefaction in the brain has been shown in rodentswith cardiovascular risk factors, such as hypertension, diabetes, andobesity. Aβ depositions are present in the neurovascular unit and their presenceis associated with microvascular impairment, which contributes to thedevelopment of early-stage pre-plaque cognitive dysfunction as well assubsequent progression of the disease.71 Furthermore, increaseddeposition of Aβ results in degeneration and disappearance of capillaries andsmall vessels, i.e. microvascular rarefaction.73 Subsequently, capillaryloss leads to an impaired clearance of Aβ, which further promotes vascular damage. Aβ accumulations on capillaries may contribute to the reduced braincapillary density via endothelial apoptosis due to its toxic effects,76 and viaits intrinsic anti-angiogenic activity and capacity to bind other pro-angiogenic factors such as VEGF. Several studies have revealed a decreased microvascular density and anincreased number of string vessels in the brain of AD patients.7985AD, a neurodegenerative brain disease, is the most common cause of dementia inthe elderly. Although deposition of amyloid β (Aβ) and hyperphosphorylatedmisfolded tau have long been the focus in explaining the molecular cause of AD,accumulating evidence now suggests that the early stage of AD might be primarilya microvascular disorder.7987 Although microvasculardensity is lower in younger AD and cSVD cases in comparison with age matchedcontrols, microvascular density in these patient groups and controls convergesby the ninth decade of life.62 Since microvasculardensity also decreases with age in absence of disease, it can be challenging todistinguish the effect of ageing from disease specific processes in cSVD andneurodegeneration.Ageing may not be considered a pathology, but its processes show considerableoverlap with cSVD and neurodegeneration. Numerous studies have shown a decreasedcerebral microvascular density in the brain of healthy aging humans. Although the etiology of cognitive deficits post whole brain radiationtherapy remains unclear, microvascular rarefaction appears to be an importantcontributor. Indeed, whole brain radiation therapy induced profoundmicrovascular rarefaction in rats and mice,90 and a reduced capillarydensity in the hippocampus of these mice is linked to deficits in learning and memory. It has been shown that brain irradiation induces dose-dependentendothelial apoptosis, loss of pericytes, and impaired angiogenesis, all promoting rarefaction. Therapeutic strategies, such as chronicsystemic hypoxia, restore capillary density and this results in the rescue ofcognitive function.Whole brain radiation therapy leads to cognitive impairment in 40-50% of braintumor survivors.9496 Inconjunction with this, studies have not only revealed a decreased cerebral bloodflow (CBF) in WMH, but also in undamaged brain regions.97 Likewise, a CBF reduction wasalready detectable before any brain lesions appeared. These findings support a causal role of hypoperfusion rather than a secondaryreduction of perfusion due to a reduced metabolic demand of damaged tissue in thebrain.During ageing and disease progression, both brain capillaries and neural tissue arelost. Whereas loss of neural tissue could result in a decreased need for oxygen andglucose and subsequently might lead to capillary loss, several studies have shown anincreased oxygen extraction from the blood in WMH, suggesting capillary loss occursfirst.This section explains and discusses advanced MRI techniques, which can providemeasures that reflect certain aspects of the pathophysiological pathways associatedwith microvascular rarefaction in human. Individual capillaries cannot be visualizedmorphologically and the various pathophysiological processes cannot be visualizeddirectly. Instead, MRI can give a proxy by measuring their functionalcharacteristics such as microvascular volume and flow, wall permeability,cerebrovascular reserve capacity, and also sense various sizes of small bloodvessels. These functional MRI measures do not reflect properties of individualmicrovessels, but the combined effect of a large number of microvessels in a tissueregion with a size of one or a few cubic millimeters. A summary of the discussed MRItechniques is provided in Dynamic susceptibility contrast-enhanced (DSC) MRI is a technique that measureshemodynamic characteristics, such as cerebral blood flow and volume. In the DSCMRI method, multiple spin echo (SE: T2-weighted) or gradient echo (GE:T2*-weighted) images are acquired with high temporal resolution during anintravenous bolus injection of a paramagnetic contrast agent (e.g. gadolinium).This dynamic measurement typically only captures the first passes of thecontrast agent through the brain. The contrast agent particles change the localhomogeneity of the magnetic field (cause T2 and T2* shortening) inside and inthe vicinity of the blood vessels, thereby lowering the signal intensity. Theamount of this signal attenuation is related to the contrast agentconcentration, which enables converting the measured signal intensity curves toconcentration curves over time. Subsequently, the quantification of CBF,cerebral blood volume (CBV) and mean transit time (MTT) of the contrast agentthrough the brain tissue can be obtained by applying a computational hemodynamicmodel to the tissue and arterial concentration curves, which also corrects forthe blood supply. A more advanced DSC measure worth mentioning is the capillarytransit time heterogeneity (CTTH), which reflects whether the capillary networkis perfused homogenously. Though DSC-MRI has been well established in clinical research, theprocess to obtain quantitative measures is complex and a thorough understandingof its acquisition parameters, image processing and limitations is needed toensure accurate results. Most of these aspects concerning DSC-MRI havepreviously been reviewed.DSC-MRI is widely used in clinical research, since it provides a strong signaleffect between the pre- and post-contrast signal and has a short acquisitiontime (2-3 min).100 Accordingly, SE isfound to be more representative when measuring microvascular perfusioncharacteristics compared to GE, and thus is the favorable technique to detect alterations in capillaryperfusion as a proxy for microvascular rarefaction. For example, the relativeCTTH measured by SE was found to correlate with WMH load and cognitiveimpairment in an AD cohort, while these same correlations could not be shownwhen CTTH was measured by GE. However, due to the stronger signal modifying effect GE has previouslybeen more frequently performed than SE. Using GE, hypoperfusion has beendemonstrated in AD patients compared to controls.104 In contrast, however,few studies found hyperperfusion in prodromal AD (at the stage of mild cognitiveimpairment), which was hypothesized to be a compensatory mechanism.104Furthermore, a strong negative correlation was found between brain perfusionmeasured with GE and cSVD-burden score. An example of a CBF map measured with DSC MRI in cSVD is shown in GE is different from SE sequences with respect to their sensitivity for vesselsize. GE shows a stronger signal drop, since the GE signal is effected by thecontrast agent in blood vessels of all sizes, whereas the SE signal is mostsensitive to magnetic field inhomogeneities in and around the small bloodvessels (radius <10 μm). Two measurement approaches can be distinguished in vessel size imaging:steady state imaging and dynamic imaging.Vessel size imaging is an MRI technique that relies on measuring both a SE (forrelaxation rate R2 = 1/T2) and GE (for R2* = 1/T2*) before and after contrastagent administration. Since SE is more sensitive for small vessels than GE, theratio of changes in relaxation rates ΔR2 and ΔR2* is dependent on the densityand the average radius of the small blood vessels, which can be calculatedwithin a voxel by this technique.The steady state approach acquires only two R2* and R2 measurements (one pre- andone post-contrast). During the post-contrast measurement, the contrast agentconcentration in the blood needs to be stable, which is the reason thatultra-small paramagnetic iron oxide (USPIO) is a more suitable contrast agentfor the steady state approach than gadolinium-based contrast agents (USPIOs havea slower clearance). The dynamic approach acquires high temporal SE and GEimages before, during and after the contrast agent concentration peak.Hereafter, the pre- and post-contrast relaxation rates are calculated to obtainΔR2* and ΔR2 curves over time. The dynamic approach is preferred in humanresearch, since gadolinium based contrast agents have considerably less healthrisks than USPIOs and the acquisition period can be kept shorter. Furthermore, we would like to point out that the fast dynamic (hybrid)GE-SE sequence can also produce CBV and CBF maps, assuming that the temporalresolution remains sufficient. Therefore, this technique can easily replace DSC-MRI.The measures that can be determined using vessel size imaging are the mean vesseldensity (Q), the mean vessel diameter (mVD) and the vessel size index (VSI) orvessel size radius. Q and mVD are related to the vessel architecture, however,they are also dependent on the acquisition parameters and on the amount of waterdiffusion in the brain, since the amount of parenchymal water diffusion isrelated to the amount of signal attenuation of the spin echo. The VSI, expressedin μm, is a diffusion-corrected measure, hence an additional MRI acquisitionshould be performed to determine the tissue diffusion coefficient.Recommendations for vessel size imaging parameters and analysis methods areexplained elsewhere.109 Vessel size imaging hasalso recently been performed in patients with subcortical vascular dementia. In the white matter of these patients, a higher mVD and VSI was foundcompared to healthy controls, probably due to dilatation of small vessels and/ora relatively fast decreasing number of small vessels in dementia. Takentogether, vessel size imaging seems to be a promising technique to revealinformation about the microvascular architecture in-vivo, although it has notyet been investigated extensively in diseases associated with rarefaction.Up to this point, human vessel size imaging research was mainly performed inpatients with brain tumors, where two studies have demonstrated that the VSI oftumor tissue highly correlated with the true vessel radius, obtained byhistopathology.The integrity of the cerebral microvascular walls can be measured with dynamiccontrast-enhanced (DCE) MRI. This technique acquires dynamic T1-weighted imagesbefore, during and after gadolinium-based contrast agent administration. Thegadolinium molecules lower the longitudinal relaxation time (T1), and thereforeenhance the T1-weighted signal. Recommendations for DCE MRI acquisition parameters and analysis forsubtle BBB impairment (e.g. in cSVD and AD), which differ from those used forhigh BBB leakage rates (e.g. in high-grade tumors and stroke lesions), weregiven elsewhere. For subtle BBB leakage, the simple pharmacokinetic graphical Patlakmethod is recommended, which neglects the reflux of contrast agent. The mainoutput measure is the BBB leakage rate transK, whichis the product of the vessel surface area (S) and the permeability (P). An additional output measure of this model is the blood volume fraction(bv).When the blood-brain barrier (BBB) is disrupted, contrast agent particles are nolonger fully restricted to the vascular compartment and can leak into theextravascular extracellular space of the brain tissue . RetentitransK, and the longscanning time (>15 minutes) to allow the contrast agent particles toextravasate from the blood into the tissue.Although measuring BBB leakage with DCE MRI is promising, it has some limitationsthat impede the use of this technique in routine clinical practice. The mainlimitations are the significant image noise influencing the tissue concentrationcurves and consequently 115 andearly AD. In cSVD, it has been demonstrated that a higher BBB leakage rate(measured with DCE-MRI) is correlated with decreased perfusion (measured with DSC-MRI), and with decreased cognitive performance. The BBB was also found to be increasingly impaired with  aging.DCE-MRI has previously revealed an increased BBB leakage rate in cSVD Arterial spin labeling (ASL) can measure cerebral blood flow (CBF) by usinginflowing water as an endogenous tracer. This technique inverts themagnetization of the blood water spins in the feeding arteries, thereby labelingthe blood before it enters the brain. The brain tissue is imaged after a delaytime, which allows the labeled spins to travel from the arterial compartment tothe tissue capillary bed. For each labeled image, a corresponding control imageis acquired. After image acquisition, the difference is calculated between thenon-labeled and the labeled signal, which is proportional to the CBF. In pCASL, a train of short RF pulses is applied to invert the spinsflowing through a labeling plane that is positioned orthogonally to the artery is most commonly used in clinical research applications.e artery. Thelen However, ASL has a lower contrast effect compared to DSC-MRI. Thecontrast effect of ASL is particularly low in white matter, since white matterhas a longer ATT and lower perfusion compared to grey matter. Therefore, performing ASL at a magnetic field of 3 T or higher isstrongly preferred, as this increases the contrast-to-noise ratio. Although ASL is ready for clinical use, new variants are still emergingto address the remaining challenges and to provide additional physiological measures, such as the rate ofwater exchange across the BBB.ASL is an acknowledged MRI technique to measure CBF quantitatively in the greymatter. Consensus recommendations have been given for its clinical implementation. Note that CVR can also be measured using other MRI techniques, forinstance based on the blood-oxygen-level-dependent (BOLD) effect, which werereviewed elsewehere.Since ASL is non-invasive and can easily be repeated, this technique is wellsuited to measure another relevant biomarker reflecting the microvascularcondition: cerebrovascular reserve (CVR). To determine CVR, CBF is measured inboth resting state and maximum flow state .This maximum flow state can be created physiologically  or pharmaceutically (acetazolamide administration). The CVRdescribes to what extent the CBF is able to increase during the vasodilatorchallenge compared to baseline. In patients with structural rarefaction, mostresidual capillaries are already perfused and dilated at rest, so they lack theability to further increase their CBF in critical situations. The hypoperfusion patterns found with ASL resembled fluorodeoxyglucosepositron emission tomography (FDG-PET) derived hypometabolism patterns in AD andmild cognitive impairment. Furthermore, ASL demonstrated a decreased CBF in cSVD within WMHs, and perilesional zones, as well as in healthy aging. Nevertheless, CBF measurements by ASL should be interpreted with cautionin these populations, since the measured hypoperfusion could have partially beencaused by a longer ATT and thus might not entirely reflect functional rarefaction. One advanced ASL technique worth considering in cerebrovascular diseasesis multi-delay ASL, which corrects the CBF for ATT at the cost of longer imagingduration or fewer label-control pairs (reducing the contrast effect) and morecomplex imaging processing. When multi-delay ASL is not feasible in populations with expectedprolonged ATT, an ASL with long PLD is advised, including those whererarefaction is likely expressing.Previous ASL studies in healthy aging and AD have critically been reviewed,whereby it was concluded that ASL is a promising tool to measure hypoperfusionin AD, contributing to early AD diagnosis. When capillaries are assumed to follow a random pattern of directions,the flow of blood molecules through the capillary network can be referred to aspseudo-diffusion. The microvascular pseudo-diffusion is typically ten timeshigher than the parenchymal diffusivity. IVIM parameters can be obtained by fitting a biexponential model,resulting in the parenchymal diffusion (fD*) is assumed to represent a proxy of themicrovascular blood flow, though physical units differ. Examples of Intravoxel incoherent motion (IVIM) imaging is a diffusion weighted imagingtechnique that can provide information about microvascular perfusion, inaddition to the parenchymal diffusivity. The IVIM perfusion measures (fD*) were reduced in the hyperventilationcondition, which was in agreement with hypotheses and with the measured ASL perfusion.The ability of IVIM to measure perfusion was validated by a study scanninghealthy volunteers during normal breathing as well as hyperventilation. IVIM perfusion has previously been measured in cSVD, type 2 diabetes, AD 139 and neurologically normal aging subjects. These studies anticipated to find a reduced blood flow volume and/orhypoperfusion (lower fD*), yet instead they found an increasein fD* in the diseased/older group comparedto the control group. These studies reasoned that the small blood vessels weredilated or that  Other factors may increase the attenuation of the microvascular componentin IVIM and thus cause 142 BBBleakage,143 and vessel tortuosity. Since rarefaction is often linked with one or more of these vascularalterations, the IVIM perfusion measures  are rather indicative ofmicrovascular integrity than mere CBF.Measuring acute hypoperfusion with IVIM was shown to be feasible in strokepatients, whereas the interpretation of the IVIM perfusion measurement inpatient groups with chronic hypoperfusion is not straightforward. PET can also be used to measure subtle BBB leakage. Although these other techniques can have specific advantages, they are lesswidely applied than MRI, as MRI provides detailed information on the brain’s anatomyand tissue lesions in the same examination session, can be more easily repeated overtime, and involves no ionizing radiation exposure or administration of iodinatedcontrast material. Since microvascular rarefaction is assumed to be a systemicprocess, measures of rarefaction in other, more easily accessible, organs couldserve as a surrogate marker for cerebral microvascular rarefaction. Especiallymeasures of microvascular density in the retina, obtained by optical coherencetomography angiography, seem to be suited as surrogate marker due to the anatomicaland functional similarity of the cerebral and retinal vessels. Other commonly used techniques to measure microvascular rarefaction includesublingual and skin intravital video-microscopy, and nailfold capillary microscopy.An extensive description of these non-MRI techniques is beyond the scope of thisreview.In this review we focused on MRI for the assessment of microvascular rarefactionsince we believe it is the most feasible and versatile non-invasive imaging methodto measure cerebral microvascular rarefaction in human in vivo. Other imagingmodalities that can be used to measure CBF in human include positron emissiontomography (PET), single photon-emission computed tomography (SPECT), and computedtomography (CT).Considering the growing ageing population, microvascular related brain disorders suchas AD and VCI will become an even bigger worldwide health issue in the future thenthey already are today. Lack of early diagnosis and effective treatments are majorproblems. To enable early diagnosis and to find targets for new therapies, a betterunderstanding of the underlying pathophysiological pathways leading to thesemicrovascular brain disorders is needed.In this review we have pointed out four main pathophysiological pathways involved inmicrovascular rarefaction , andtheir possible interplay. Although the pathophysiological pathways underlyingmicrovascular rarefaction in the brain are becoming clearer, there are still anumber of knowledge gaps to be filled in. Currently, most research into thepathophysiology of microvascular rarefaction has been conducted in animal models,whereas studies in humans are needed to confirm that findings in animals relate tothese in human. More research investigating the different pathophysiologicalprocesses simultaneously is needed to learn more about the sequence and the relativeimportance of these processes. Furthermore, little is known about the relation ofmicrovascular rarefaction with other processes known to play a role in microvascularrelated brain disorders .transK obtained with DCE-MRI. The interpretationof transK (P · S) is not straightforward in the contextof microvascular rarefaction. On one hand, a reduced capillary density leads to areduced total vessel surface area S, contributing to a decreased leakagetransK. On the other hand, pericyte loss andendothelial apoptosis result in a leaky vessel wall, causing an increase inpermeability P, resulting in an increased transK.Previous studies have shown increased transK in diseasesassociated with rarefaction,114116 suggesting that theincrease in P dominates. The detectable effects of vessel diameter changes inrarefaction by vessel size imaging are also debatable. The effects of endothelialdysfunction (i.e. reduced arteriolar vasodilator capacity) and enhanced pericytecontraction would lead to a decreased vessel radius. Meanwhile, there are othereffects causing an enlarged mean vessel radius, e.g. vessel dilation as acompensatory mechanism for the reduced CBF in structural rarefaction or as a resultof pericyte loss, and the occlusion or collapse of the smallest capillaries. Which techniques serve best to measure microvascular rarefaction at whatstage of the pathological cascade in microvascular brain disorders is likely acomplex puzzle to be solved.Existing MRI techniques can increase the knowledge about the role of microvascularrarefaction and its underlying pathophysiological pathways in microvascular braindisorders. The various techniques described in this review cannot depictmicrovessels in a morphological way, but can measure various functional aspects ofmicrovascular rarefaction.  However, contrast agents have a small risk to induce contrast nephropathy inpatients with pre-existing renal impairment. The endogenous contrast agent based MRItechniques (i.e. ASL and IVIM) can be used as an alternative in people withcontraindications for contrast agent, and are preferable when multiple measurementsover time are required. The endogenous techniques are still emerging, and a detailedunderstanding of these techniques is needed for clear interpretation of theperfusion MRI measures in microvascular brain disorders. Researchers should be awareof the confounding factors, i.e. the influence of the arterial transit time on ASLperfusion signal and the non-perfusion factors that contribute to the IVIM perfusionsignal.In the context of microvascular brain disorders, the physiological MRI techniquesdescribed are mainly used in clinical research settings. These techniques can beclassified into exogenous and endogenous contrast agent based techniques. Ingeneral, the strength of exogenous contrast agent based MRI techniques  is the strong contrast effect.Furthermore, the image acquisition and analysis protocols for advanced MRI techniquesvary widely, which hampers progression in understanding the underlyingpathophysiological pathways in microvascular rarefaction and its contribution to theclinical features of microvascular brain disorders such as AD and cSVD. To enablecomparing studies over various sites, MRI protocols for image acquisition and imageanalysis need to be standardized. Furthermore, there remains a need for validation of these MRI techniques incombined radiological-histological studies to confirm that the obtained MRI measurescorrelate with actual structural microvascular rarefaction. The internationalCRUCIAL consortium  aims to identify cerebral microvascular rarefaction using advanced MRItechniques in a population of VCI patients and healthy aged controls, and willvalidate these MRI techniques by scanning and performing histologic examination onrat brains.More studies investigating these MRI techniques in human microvascular braindisorders are essential to establish test-retest repeatability and for validation ofsensitivity to the subtle changes which can be found in these disorders. Ideally,longitudinal studies are needed to assess true rarefaction, i.e. temporal changes inmicrovascular density throughout disease progression and during healthy aging.Considering younger subjects are more likely to show significant differences invessel density due to pathologies such as AD and cSVD in comparison with agedmatched controls, it might be valuable to prevent oversampling of oldest-oldsubjects in future studies.We reviewed the evidence for microvascular rarefaction as an early pathophysiologicalprocess in a number of microvascular brain disorders and normal ageing, and thevalue of advanced MRI techniques addressing different properties of themicrovasculature for its assessment. Although these MRI techniques are promising tocontribute to early diagnostic information and to be used for monitoring treatmentresponse in microvascular brain disorders in the future, these techniques are stillemerging and do not provide disease specific information. More clinical studies,preferably longitudinal, are needed in large patient populations with various braindisorders as well as in aging.


Molecular carcinogenicity is a preventable cause of cancer, but systematically identifying carcinogenic compounds, which involves performing experiments on animal models, is expensive, time consuming and low throughput. As a result, carcinogenicity information is limited and building data-driven models with good prediction accuracy remains a major challenge.In this work, we propose CONCERTO, a deep learning model that uses a graph transformer in conjunction with a molecular fingerprint representation for carcinogenicity prediction from molecular structure. Special efforts have been made to overcome the data size constraint, such as multi-round pre-training on related but lower quality mutagenicity data, and transfer learning from a large self-supervised model. Extensive experiments demonstrate that our model performs well and can generalize to external validation sets. CONCERTO could be useful for guiding future carcinogenicity experiments and provide insight into the molecular basis of carcinogenicity.https://github.com/bowang-lab/CONCERTOThe code and data underlying this article are available on github at  In silico predictions to assess carcinogenicity provide an appealing alternative, and can help select compounds for costly downstream analysis.Globally, cancer is the second leading cause of death, and accurate molecule carcinogenicity prediction holds promise in decreasing the likelihood of disease onset . Cancer Traditionally, carcinogenic compounds have been identified as a result of observational studies in sub-populations with increased cancer penetrance. This approach is effective in two scenarios: the first is if the compound is extremely carcinogenic, e.g. aristolochic acid, which was present in certain herbal supplements before being identified as one of the most potent compounds in the carcinogenic potency database (CPDB) . These aCurrent state-of-the-art solutions for molecule carcinogenicity prediction train models using ASCII string representations of molecules through simplified molecular-input line-entry systems (SMILES) or molecular fingerprints, hand engineered features capturing core molecular properties . The node and edge features depend on the choice of model, but include important chemical properties like atomic number, atomic mass and bond order. The second, fingerprint-based models use hand engineered features that aim to summarize important molecular properties like molecule aromaticity, presence of functional groups and atom co-occurrence . FingerpFundamental technical challenges at the core of the carcinogenicity prediction problem are the difficulty of data acquisition, and label representation. Every experiment is conducted in animals, usually rodents, resulting in dozens of animals required for a single data point. Not only does this result in limited training data (approximately 1000 unique compounds), but presents difficulties for robust model evaluation. Previous methods have treated this problem as binary classification task which results in loss of information regarding the degree of compound potency (The CONCERTO architecture consists of a large self-supervised GNN transformer and multilayer perceptron (MLP) optimized over a molecular fingerprint representation concatenated with GNN transformer representation . It is t3.2.1Given of a set of nodes (or vertices) G as input in the form of an adjacency matrix and use node-wise and/or edge-wise layer embeddings to learn a non-linear predictive mapping. MPNNs are a specific type of GNN which aggregate information in the form of ‘messages’ across neighborhoods of respective nodes   resulting in noisy measurements with lower rates of reproducibility . AlthougFor mutagenicity pre-training and evaluation we use a dataset generated by 3.2.4large embedding in addition to 2048 dimensional Morgan, RDKit and MACCS fingerprints. The MLP consists of five layers each containing dropout with probability 0.1, batch normalization and a ReLU activation function , and d is the administered dose-rate of a respective test molecule . The estimated TD50 value is then calculated as β. We transform the calculation for TD50 to For model training we utilize CPDB, which is a collection of 6540 experimental tests containing results from long-term carcinogenesis bioassays, primarily in rodents, for over 1000 chemical compounds  Gold et:(3)λ(t,For external test evaluation we use CCRIS, which is a database containing experimental test results of over 4500 chemical compounds gathered from various studies cited in literature (In this work, we introduce a new dataset for carcinogenicity analysis and use a perturbation approach for evaluating functional group importance both of which require measuring molecular distances. To measure similarity between individual molecules, we compute Tanimoto similarity of their molecular fingerprints. This pairwise measure allows us to estimate dataset variance, using the diversity metric, and distance between dataset distributions, using maximum mean discrepancy (MMD; fph is a mapping from SMILES strings to vectorized binary representations. Then, we define the Tanimoto similarity coefficient asTo calculate Tanimoto similarity, between two molecules erprints  as x=hfpx and y are binary vectors, Since We make use of the MMD score to define a distance metric between molecular datasets. Given two sets of molecular fingerprints that are sampled from two distributions We use the Tanimoto similarity coefficient as the kernel function, i.e. We perform hyperparameter searches over CONCERTO architectures, fingerprint MLP and GROVER MLP, and find that CONCERTO outperforms the standalone models on both test sets . StandalThe full CONCERTO model consists of GROVER embeddings, fingerprint representations, trained using alternating carcinogenicity and mutagenicity objectives. We perform ablation experiments to identify contributions of individual components. We conduct 50 runs with matched seeds on a well performing set of hyperparameters to evaluate whether addition of pre-training improves performance on CPDB and CCRIS. We find that providing GROVER representation to the models improves performance on both the test sets . The mutWe utilize Exmol to better understand our model’s behavior by identifying molecular substructures that are important drivers in carcinogenicity prediction (We extend the aforementioned analysis by comparing the frequencies of known toxicophores found in the test data with a corresponding set of counterfactual examples. Our results suggest that CONCERTO learns carcinogenic functional groups. For every molecule in our test set, we generate a positive carcinogenic counterfactual and a negative non-carcinogenic counterfactual. We then check the generated molecular structures for the presence of known toxicophores  (Kazius To better understand differences between our datasets, we calculate maximum mean discrepancy over Tanimoto scores, as described in Predicting molecular carcinogenicity is an important public health problem to address, but due to the prohibitive cost and difficulty of measuring carcinogenicity many compounds lack experimental data. In this work, we investigate three orthogonal approaches to overcome dataset size constraints: architecture choice, dataset modification and pre-training techniques. First, we leverage the inductive bias present in GNN architectures relating to the graphical molecular structure. We find that GROVER, in conjunction with an MLP-fingerprint predictor outperforms the fingerprint MLP model as well as the previous state-of-the-art model. We suspect that due to the limited size of available data, a combination of transfer learning from a large GNN, and hand-engineered features extracted from molecular structures, is effective at capturing important drivers of molecular carcinogenicity. Next, we augment the dataset with more informative labels by aggregating individual experimental results and creating continuous labels. This creates a richer representation for the network and circumvents the MTD design problem, where a molecule could be carcinogenic at a maximum dose for the animal but it would be impossible to be exposed to that dosage in the natural world. In addition, we collect an external dataset five times larger than previous, allowing us to make meaningful model performance comparisons while decreasing concern of overfitting to the test set. Finally, we explore the utility of model pre-training in two forms: first utilizing transfer learning from GROVER, and second multi-round pre-training on related but lower quality experiments. The differentiable nature of our model allows us to make use of effective pre-training strategies. We assess the contributing effects of architecture choice and pre-training techniques through a series of ablation experiments see , throughAlthough compound carcinogenicity pre-screening is not a solved problem, we hope that CONCERTO will aid in selecting molecules for downstream carcinogenicity experiments due to its improved predictive accuracy over existing approaches. Given that up to 13% of recent drug retractions have been due to molecular DNA reactivity, a method for identifying functionally similar molecules but with decreased carcinogenicity could be useful . We aim In this work, we present a GNN transformer model for predicting molecule carcinogenicity. We develop a novel multi-round pre-training methodology that leverages mutagenic data to improve accuracy on the carcinogenicity task. We find that the combination of these architecture improvements and novel training techniques results in a model that outperforms the previous state-of-the-art in predicting maximum tolerable dose. Additionally, we employ counterfactual analysis to investigate model interpretability and confirm that our model recovers previous knowledge about toxicophores.


Cancer-related anemia (CRA) is a common multifactorial disorder that adversely affects the quality of life and overall prognosis in patients with cancer. Safety concerns associated with the most common CRA treatment options, including intravenous iron therapy and erythropoietic-stimulating agents, have often resulted in no or suboptimal anemia management for many cancer patients. Chronic anemia creates a vital need to restore normal erythropoietic output and therefore activates the mechanisms of stress erythropoiesis (SE). A growing body of evidence demonstrates that bone morphogenetic protein 4 (BMP4) signaling, along with glucocorticoids, erythropoietin, stem cell factor, growth differentiation factor 15 (GDF15) and hypoxia-inducible factors, plays a pivotal role in SE. Nevertheless, a chronic state of SE may lead to ineffective erythropoiesis, characterized by the expansion of erythroid progenitor pool, that largely fails to differentiate and give rise to mature red blood cells, further aggravating CRA. In this review, we summarize the current state of knowledge on the emerging roles for stress erythroid progenitors and activated SE pathways in tumor progression, highlighting the urgent need to suppress ineffective erythropoiesis in cancer patients and develop an optimal treatment strategy as well as a personalized approach to CRA management. Anemia commonly occurs in patients with cancer , either as a tumor-driven blood disorder or as a consequence of a patient’s chemotherapy or progressive disease . The preCurrent treatment options for CRA include erythropoietin-stimulating agents (ESA), iron therapy, and red blood cell (RBC) transfusions. The transfusion of RBC rapidly increases hemoglobin concentration and improves functional status in cancer patients . DespiteSimilar to anemia of other chronic conditions, CRA is mostly referred to as the anemia of inflammation . SeveralBone morphogenic protein 4 (BMP4), EPO, stem cell factor (SCF) and growth/differentiation factor-15 (GDF15) have emerged as the key players in SE. Apart from regulating SE, these factors also play roles in erythroid development during fetal and/or adult period. Thus, BMP4 is essential for the expansion of erythroid progenitors during fetal development. Unlike bone marrow progenitors, fetal liver progenitor cells can give rise to early erythroid progenitors in the presence of EPO alone, without any additional signals, reflecting the differences between adult bone marrow and fetal liver erythropoiesis . The expErythropoiesis is a well-orchestrated and tightly regulated process for maintaining a steady number of RBC and thus providing adequate oxygen delivery to tissues. Since RBC are critically important for survival, erythropoiesis during development occurs in two distinct forms. The first is termed “primitive” form and refers to nucleated erythroid cells that originate extraembryonically in the yolk sac. Apart from being essential for the oxygen delivery to cells within the rapidly growing embryo, these large nucleated erythroid cells also seem to play an important role in angiogenesis . The secIn contrast to the embryonic or fetal period, erythropoiesis in healthy adults is primarily homeostatic and occurs in the bone marrow. Under steady-state conditions, RBC are formed continuously and mature within the specialized microenvironmental niches, known as erythroblastic islands . The seqSteady-state erythropoiesis is critically regulated by the kidney-derived hormone—EPO. EPO promotes the survival, proliferation and differentiation of erythroid progenitors, primarily CFU-E cells. Binding of EPO to the EPOR on the surface of erythroid cells upregulates the expression of its own receptor, which increases EPO sensitivity as well as the expression of erythroid-specific transcription factors, such as GATA1 and KLF1, and other erythroid-specific genes (glycophorin and globin genes). In addition to EPO, BFU-E cells respond to many cytokines and growth factors including interleukin-3, SCF, insulin like growth factor-1 and glucocorticoids . During Of paramount importance for erythropoiesis is to adjust the rate of RBC production to meet physiological demands. Hence, steady-state erythropoiesis generates new RBC at a constant rate, sufficient to replace the ones that are being destroyed. However, there are times of great erythropoietic need, caused either by prolonged or severe anemia, along the time when steady-state erythropoiesis is impaired or inhibited. At these times, homeostatic erythropoiesis in the bone marrow is not sufficient to support adequate RBC production, and therefore mechanisms of SE are initiated .The current understanding of SE relies mainly on data gathered from mouse studies and experimental data have demonstrated that SE utilizes progenitor cells and signals distinct from those in bone marrow during steady-state erythropoiesis. The regulation of erythropoiesis is largely dependent on microenvironmental cues, and unlike steady-state erythropoiesis in the bone marrow, SE occurs predominantly in the mouse spleen . The proErythroid progenitors are decisive in determining the overall red cell mass and the findings derived from mouse studies have revealed that the characteristics of these cells, as well as the mechanisms that drive their extensive expansion during SE in many ways differ from steady state erythropoiesis and resemble those involved in erythropoiesis during embryonic and fetal development . It is nBMP4 gene  superfamily and contains over 20 members with a wide variety of functions (Bone morphogenetic proteins (BMPs) are signaling molecules well-known for their essential roles during vertebrate development. The BMP family belongs to the transforming growth factor unctions . Based ounctions . The actunctions . In the unctions . Besidesunctions .in vivo settings do not necessarily reflect just BMP4 activity. Additional studies on the BMP4/BMPR signaling pathway in a large number of different primary cells/cell lines from each tumor type are further needed to clarify the exact contribution of BMP4 signaling to tumorigenesis and metastasis in a tissue-specific manner  has been demonstrated in cancer cells . SeveralTaken together, due to the widespread use of nonspecific anti-EPOR antibodies that may provide false positive staining, the tumor-promoting effects of the EPO/EPOR axis have remained an outstanding issue. Further studies are needed to address this issue. In addition to antibody issues, the specific tumor biology should always be carefully considered in the interpretation of these results.via c-Kit is required for the maintenance of hematopoietic stem/progenitor cells and their interactions with niche cells , GDF15 plays a prominent role in energy homeostasis . Yang etBoth a chronic inflammatory state and increased GFD15 levels are commonly associated with cancer . Thus, tAnemia is generally recognized as an independent predictor of poor prognosis in patients with cancer. Nevertheless, the biological mechanisms underlying this relationship are still not completely understood and need to be further investigated. Thus, CRA may activate SE pathways, causing a marked augmentation of the erythroid progenitor pool in these patients, and therefore requires special attention. In particular, a growing body of evidence suggests a significant role for both stress erythroid progenitors and activated SE pathways in cancer progression. Despite the activation of SE pathways and the consequent expansion of nucleated erythroid cells, if left untreated, CRA still persists due to dysregulated/ineffective erythropoiesis in cancer patients. Inhibiting these pathways may be a promising therapeutic strategy to reduce the tumor-promoting activity of nucleated erythroid cells, along with the management of CRA. Furthermore, some of key players in SE, such as SCF/c-kit and GDF15, have been shown to modulate the erythroid cell response to differentiation stimuli and counteract erythroid cell maturation . Hence,


Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the host system. Many materials that are used in biomedical and food packaging industries are prepared by chemical means of crosslinking the proteins, besides the physical or enzymatic means of crosslinking. Such chemical methods utilize the chemical compounds or crosslinkers available from natural sources or synthetically generated with the ability to form covalent/non-covalent bonds with proteins. Such linkages are possible with chemicals like carbodiimides/epoxides, while photo-induced novel chemical crosslinkers are also available. In this review, we have discussed different protein crosslinking strategies under chemical methods, along with the corresponding crosslinking reactions/conditions, material properties and significant applications. Protein crosslinking  that resIn recent times, novel crosslinkers or crosslinking strategies have been developed as one of the promising research areas, focusing on the utilization of versatile protein biomolecules with potential biomedical and industrial applications . The majChemical crosslinking involves the utilization of different natural and/or synthetic crosslinkers to form the linkages. Protein crosslinkers usually possess reactive sites that target functional moieties like thiol, amine and carboxylic acid groups in the side chains of amino acid residues. The chemical crosslinkers can further be classified under two groups, namely, non-zero length crosslinkers and zero length crosslinkers ,31. Non-The crosslinkers or chemical compounds obtained from natural sources like plants are categorized as natural crosslinkers. The commonly used natural crosslinkers are as follows:Gardenia jasmonoides Ellis  propanoic acid (THPP) was initially reported as a crosslinker for the generation of elastin-like protein materials ,102. ThiThe SpyTag/SpyCatcher system was developed by a covalent reaction between the protein partners acquired from the fibronectin-binding protein (FbaB) of Streptococcus pyogenes . Here thThe chemical crosslinking methods using synthetic crosslinkers mentioned above, along with significant applications, are summarized in There are several other crosslinkers that find potential applications as bioconjugates, target/ functional group-specific linkers, nano-particle preparation, etc. Recently several novel maleimides that are rationally designed and are known to overcome the shortcomings of classical maleimides have been reported in the literature. The very good reactivity and synthetic accessibility offered by these maleimides towards bioconjugation make them powerful tools for protein crosslinking, particularly for drug conjugation to antibodies. The mechanism of crosslinking proteins using maleimide esters is depicted in Similarly, numerous photo affinity probes have also been recently reported and are known to be involved in protein-protein interactions. A bifunctional amino acid containing a terminal alkyne group and a diazirine as a photo-crosslinker was utilized for bio-orthogonal tagging (A bifunctional amino acid to study protein–protein interaction . The mosHerein we have discussed various chemical methods of protein crosslinking. The applications of these strategies or methods to generate crosslinked protein networks or scaffolds, along with the limitations of each, are reviewed in detail, along with recent literature updates. The implementations of the generated biomaterials for applications in biomedical and food packaging industries are discussed, along with their advantages and limitations. A comparison of the toxicity generated and strength exhibited by materials created using natural agents over synthetic materials is elaborated. Moreover, chemical crosslinking methods employing natural agents are used to obtain covalent crosslinking as that obtained using synthetic agents without causing toxicity to the crosslinked material/film. Several applications, including scaffold materials, biomaterials for tissue engineering, drug delivery systems/vehicles, food packaging materials, edible protein films etc., have been achieved by chemical methods of protein-based crosslinking .Protein biomolecules possessing versatile features can be exploited for multiple applications. The extent of reinforcement is very much essential and needs to be achieved with better efficiency through novel crosslinking methods that can overcome the limitations of the presently available methods that result in protein-based materials/films which are non-toxic and biodegradable. With the constant demand in the bio-medical field for various protein-based scaffolds, tissue sealants, drug carriers and other bio-prosthetics, it is necessary for researchers to focus on creating novel protein-based materials that ultimately require protein crosslinkers. Biodegradable protein materials  could seMost of the existing protein crosslinking strategies, especially chemical crosslinkers, have the possibility of causing damage to protein structure/properties or the cells/tissues in end-use. Chemical crosslinking using synthetic crosslinking agents like EDC-NHS faces the major issue of cytotoxicity, and the removal of residual crosslinking agents is crucial. Natural chemical crosslinking agents, though they have the advantage of not conferring cytotoxicity, are known to possess a lower degree of crosslinking relative to that of synthetic crosslinkers. However, there are many other protein-based crosslinkers like leucine zipper domains or SpyTag/SpyCatcher systems and natural chemical crosslinkers that reinforce the protein polymers into materials without any toxic effects have always been the interest of many researchers. Specific protein-protein interactions with naturally available chemical compounds from plants or synthetic chemicals that can act like catalysts or zero-length crosslinkers ought to be explored further for better protein crosslinking efficiencies. Massive chances are still available to develop many more novel protein-based or natural/synthetic chemical crosslinkers to be utilized for various available proteins for the betterment of humankind.


Family members often take on the caregiver roles to stroke survivors due to kinships and cultural responsibilities. Yet, little is known about the the impact of survivor-caregiver dynamics on stroke rehabilitation. To understand the dyadic relationships between the dyads, a systematic review was conducted to examine studies which have adopted Actor–Partner Interdependence Model (APIM) analysis.A systematic review and meta-analysis were conducted using the following electronic databases: PubMed, SAGE Journals, MEDLINE, PsycINFO and Cochrane Review to identify eligible studies published from their inception to December 2021, following the PRISMA guidelines.Nine studies involving 1183 stroke survivors  and 1181 caregivers  had met the inclusion criteria and were identified in this review. The interaction among the following outcomes were self-esteem, optimism, stress, depression, emotional distress, quality of life, and life satisfaction. The review provided evidence that (1) APIM is valuable tool in analyzing dyadic interactions; (2) support from caregivers has a significant impact on the stroke survivor’s recovery; (3) the mental health status of caregivers can influence that of stroke survivors and vice versa; (4) Due to the interconnected nature of dyadic interaction, providing dyadic intervention have positive impact on dyads.These findings highlight the interdependence nature between survivor-caregiver dyads in the context of stroke rehabilitation. The APIM provides conclusive evidence on the effectiveness of survivor-caregiver dyadic interactions, with significant theoretical and practice implications for both health and social care professions. More research is needed to support dyadic strategies for stroke survivors.