Tauopathy, characterized by the hyperphosphorylation and accumulation of the microtubule-associated protein tau, and the accumulation of Aβ oligomers, constitute the major pathological hallmarks of Alzheimer’s disease. However, the relationship and causal roles of these two pathological changes in neurodegeneration remain to be defined, even though they occur together or independently in several neurodegenerative diseases associated with cognitive and movement impairment. While it is widely accepted that Aβ accumulation leads to tauopathy in the late stages of the disease, it is still unknown whether tauopathy influences the formation of toxic Aβ oligomers. To address this, we generated transgenic cynomolgus monkey models expressing Tau (P301L) through lentiviral infection of monkey embryos. These monkeys developed age-dependent neurodegeneration and motor dysfunction. Additionally, we performed a stereotaxic injection of adult monkey and mouse brains to express Tau (P301L) via AAV9 infection. Importantly, we found that tauopathy resulting from embryonic transgenic Tau expression or stereotaxic brain injection of AAV-Tau selectively promoted the generation of Aβ oligomers in the monkey spinal cord. These Aβ oligomers were recognized by several antibodies to Aβ1–42 and contributed to neurodegeneration. However, the generation of Aβ oligomers was not observed in other brain regions of Tau transgenic monkeys or in the brains of mice injected with AAV9-Tau (P301L), suggesting that the generation of Aβ oligomers is species- and brain region-dependent. Our findings demonstrate for the first time that tauopathy can trigger Aβ pathology in the primate spinal cord and provide new insight into the pathogenesis and treatment of tauopathy. The neuropathological features in AD are characterized by tauopathy and the extracellular plaques composed of β-amyloid (Aβ) aggregates.8 The development of these two well-established pathological hallmarks is well correlated with the clinical disease severity,10 and it has been widely accepted that Aβ accumulation leads to tauopathy in the late stage of diseases,11 However, it has also been recognized for long time that depletion of tau is protective against Aβ-mediated deficits.15 Despite these interesting findings, it remains to be investigated whether tauopathy can act as the primary cause to trigger Aβ-related neuropathology.Microtubule-associated protein tau, which is encoded by the MAPT gene, is highly expressed in neuronal axons and participates in microtubule assembly and function of axons.MAPT gene in FTD16 has led to great efforts on the generation of transgenic animal models to elucidate the role of Tau in neurodegeneration. Transgenic Tau animal models, especially rodent models, remarkably recapitulate tauopathy and have provided important insight into the pathogenesis of tauopathy-related diseases.21 However, Tau is expressed through alternative splicing in the human brain, resulting in six isoforms containing three repeats (3 R) or four repeats (4 R), whereas only 4 R Tau is expressed in the adult rodent brain.24 It is known that tauopathy also occurs in the aged brains of non-human primates but is absent in the brains of old small animals including rodents.28 Furthermore, human Tau consists of 11 primate-specific amino acids, which can be found in macaques but not in rodents.29 There are a large number of phosphorylation sites (at least 40 serine/threonine and two tyrosine sites) in human Tau32 and considerable disparity in phenotypes of a variety of Tau transgenic animal models.33 Considerable differences exist in brain development, anatomical structure, cognitive and behavioral complexity between small and large animals. These differences explain why many human neurological and neuropsychiatric diseases are inadequately modeled in small animals. The primate brain, with its increased neuronal number and larger cerebral cortex, possesses more complex neural architectures that determine cognitive abilities. These differences can also impact the pathological and behavioral phenotypes of animal models used in neurodegenerative disease research. Aging processes also vary significantly among different animal species. Rodents, for example, typically have a lifespan of less than 3 years, which may not be sufficient for the occurrence of neurodegeneration or other important pathological changes that usually take decades to develop in humans. The rapid development and accelerated aging in small animals could make their brains or neuronal cells less vulnerable to insults or toxic proteins, resulting in a lack of robust neurodegeneration despite the presence of impaired brain function.Animal models are vitally important for investigating the pathogenesis of tauopathy. The identification of mutations in the 36 Our findings indicate that monkey models more accurately replicate neuropathology observed in patient brains and offer valuable insights into disease pathogenesis that may not be obtained from small animals. For instance, monkeys with mutations in the PINK1 gene, which can lead to early onset Parkinson’s disease, exhibit neurodegeneration, whereas PINK1 knockout mice do not display such degeneration.35 Similarly, monkeys with mutations in the CHD8 gene, which are responsible for autistic behaviors and macrocephaly in humans, exhibit abnormal gliogenesis, a phenomenon absent in mutant mouse models.36 These discoveries provide compelling evidence for the use of non-human primates as an essential model for investigating neuropathology and pathogenesis-related to AD.Therefore, it is logical and important to utilize non-human primates to investigate the role of human Tau and its related pathology, given their closer resemblance to human brain structure and function. In our early studies, we employed a CRISPR/Cas9 targeting approach to generate genetically modified monkeys for the purpose of modeling significant brain diseases.Tau P301L (0N4R P301L) via embryonic lentiviral transduction and stereotaxic brain injection of AAV. We found that mutant Tau elicited neurodegeneration accompanied by typical features of tauopathy and motor function deficits. Moreover, transgenic Tau selectively promotes the production of toxic Aβ oligomers in the spinal cord of Tau transgenic monkeys and adult monkeys injected with AAV-Tau (P301L). Our studies demonstrated for the first time that Tau can cause the formation of endogenous toxic Aβ in a brain region-dependent manner and provide pathogenic insight for how Tau is involved in AD related pathogenesis.Given the absence of any reports on the creation of transgenic Tau monkeys through embryonic transgenic expression, it is imperative to establish such a monkey model to investigate tauopathy and its relationship with Aβ accumulation. In the current study, we established non-human primate models that express mutant human 36 We injected 88 mature cynomolgus (Macaca fascicularis) oocytes with high titer (107 vg ml−1) lentiviruses expressing human MAPT gene with Tau p301L mutation (0N4R P301L) that was also tagged with the Flag epitope at the N terminus and expressed under the control of the mouse prion promoter  newborn monkeys -MRI analysis of 3 WT and 4 Tau monkeys at the age of 30 months and found that Tau transgenic monkey brains showed reduced [18F] FDG uptake to various extents 39 for Tau deposition in the monkey brains at the age of 38 months, we found that Tau deposition was more abundant in Tau monkeys than in WT monkeys  that was also evidently high in the blood sample Fig. . Howevernts Fig. , a sign eys Fig. . We perfeys Fig. , which wons Fig. . We alsoThe behaviors of Tau monkeys were compared with those of the age-matched WT monkeys under the same housing conditions. At the age of 38–42 months, we found that Tau monkeys exhibited the delayed response at 10 min in short-term memory test and at 15 and 30 days in long-term memory test Fig. . NotablyGross morphology of the Tau1 brain at the age of 37 months showed the reduced thickness of the cortex and decreased size of the caudate and putamen as compared with an age-matched WT control Fig. . WesternNeuN staining of neuronal cells showed a marked reduction of NeuN-positive cells in the ERC of Tau1 monkey  into the hippocampus and spinal cord of seven WT monkeys at young (5-6 years old) and old (25 years old) ages Fig. . For the41. However, our monkey models also showed motor impairment, which was seen in some transgenic Tau mouse models.20 In addition, there are several interesting findings that have not been reported but have important implications for pathogenesis and treatment and therefore are warranted for further discussion.Tauopathy has been found in different transgenic Tau animal models, including a recently reported rhesus monkey model that was injected with AAV-Tau into the vulnerable ERCFirst, the severe phenotypes observed in the Tau1 monkey are likely associated with its high expression of transgenic Tau, which is supported by the highest level of transgenic Tau found in its cerebrospinal fluid (CSF) and the copy numbers of transgenic Tau. The early death of the Tau1 monkey is also consistent with its severe phenotypes. Similarly, the Tau2 monkey expressed a lower level of Tau in its spinal cord compared to Tau1 and had a longer lifespan with milder phenotypes. It is possible that other Tau transgenic monkeys, which express lower levels of transgenic Tau in their brains, may develop phenotypes when the transgenic Tau accumulates at high levels in their aged brains after several years.5 Thus, although abnormally accumulated or overexpressed Tau can participate in many aspects of cellular dysfunction to cause diverse phenotypes, its high level is more likely to severely affect neuronal function related to body movement and motor activity. In support of this possibility, neuronal loss, Tau hyperphosphorylation, and tangle formation were concurrently found in the striatum and spinal cord of Tau1 monkey, which are the brain regions that critically control movement coordination and balance.Second, motor dysfunction phenotypes are more obvious and severe than cognitive deficit in Tau transgenic monkeys. The motor phenotype is in line with motor dysfunction in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick’s disease (PD) in which tauopathy is the major pathological feature.41 A critical issue is whether Aβ product we observed is an APP fragment or Aβ oligomer that is formed by Aβ1–42. To address this issue, we used multiple antibodies against different epitopes of APP and proved that it is indeed an Aβ oligomer, because it could be recognized by at least 4 antibodies for Aβ1–42 but not by those against the adjacent region or C-terminal APP.Third, the more interesting and important finding is that Tau overexpression can selectively induce Aβ oligomers in the spinal cord in monkeys, which is supported by the following evidence. One is the specific expression Aβ in the spinal cord in transgenic Tau monkeys when compared with other brain regions. Consistently, this specific accumulation is unlikely to cause increases in Aβ levels in the plasma and cerebrospinal fluid in Tau transgenic monkeys. Another is the selective Aβ accumulation in the AAV-Tau-injected spinal cord as opposed to the injected hippocampus. Thus, the fact that both embryonic transgenic and brain injection-mediated expression of transgenic mutant Tau consistently resulted in the selective expression of Aβ demonstrates a spinal cord-dependent formation of Aβ oligomer mediated by tauopathy. This finding is also supported by the lack of altered Aβ42/40 levels in the rhesus monkeys that were injected with AAV-Tau (P301L/S320F) into ERC, a brain region that projects to the hippocampus.Thus, an immediate and important issue is why only Aβ oligomer, but not monomer, dimers, and other forms of oligomers, was readily detected. We found that overexpression of mutant Tau can induce the brain region  dependent formation of endogenous Aβ oligomer in monkey, which appears to be more stable than other smaller or larger oligomers. It is likely that this particular Aβ oligomer has unique structure or conformation that enables its stability and detection by Aβ 1–42 antibodies via western blotting analysis.42 In our studies, we did not observe the production of Aβ oligomer in the cortex of transgenic Tau1 monkey. Also, because AAV-Tau-injected spinal cord in mice did not yield the same Aβ oligomer formation as seen in the monkey spinal cord, we should consider species-dependent differences in Tau-mediated production of Aβ species, which could be related to different posttranslational modifications, interacting partners, and processing of Tau.The next question is why Aβ oligomer is only induced in the spinal cord. It is possible that the spinal cord has specific microenvironment that favors the production of small Aβ1–42 while Tau accumulation induces intracellular stress that can promote the oligomerization of Aβ1–42. Another possibility is that Tau overexpression may alter metabolic or gene expression profiling to particularly lead to the generation of Aβ1–42 and its oligomer formation. Indeed, FTD P301L tau transgenic mice show oligomeric and fibrillar species of beta-amyloid (Aβ1–42) that decreased mitochondrial membrane potential in cortical brain cells.44 Since targeting Aβ monomers and plaques was unsuccessful in clinical trials,45 it appears that targeting specific forms of Aβ may be important for reducing pathology. Analyses of a large number of human brains across the lifespan have shown that tau pathology begins about a decade before the formation of Aβ plaques.47 The finding that Aβ deposition was only observed in the spinal cord of the monkey brain in the early disease stage suggests that the spinal cord might be the first site for Aβ protein accumulation. Our findings suggest that Aβ oligomer, which can be induced by mutant Tau, is stable in the spinal cord of non-human primate so that targeting this form of Aβ would be likely beneficial for treating Tau- and Aβ-induced pathology, at least in the spinal cord.Our findings show that pathological Tau accumulation can trigger Aβ oligomer formation, demonstrating for the first time that Tau can play a causal role in endogenous Aβ formation and toxicity. Although neurodegeneration in various brain regions in Tau1 monkey indicates that mutant Tau can mediate neurodegeneration without Aβ oligomer formation, the presence of Aβ oligomer in the spinal cord may generate synergistic toxicity to severely affect motor function, which may explain why Tau1 monkey displayed severe body movement difficulty. Furthermore, the toxicity of Aβ oligomer and its association with tauopathy support the importance in preventing Aβ accumulation for AD treatment and echo the recent successful clinic trial of anti-Aβ drug lecanemab, though whether targeting Aβ can effectively treat AD still remains hotly debated.M. fascicularis) were housed in cages and examined for their behaviors at Guangdong Landau Biotechnology Co. Ltd., which is an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. All animal-related protocols were approved in advance by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Landau Biotechnology Co. Ltd and Jinan University. This study occurred in strict compliance with the “Guide for the Care and Use of Laboratory Animals (2011)” to ensure the safety of personnel and animal welfare. Health and behavior of the monkeys were monitored daily by the husbandry staff and veterinarians. Animal information is presented in Supplementary information.Adult healthy female cynomolgus monkeys were housed in individual cages at Yuanxi Biotech Inc. Guangzhou and used to generate Tau transgenic monkey in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Yuanxi Biotech Inc. Guangzhou. Tau transgenic monkey and age- and gender-matched healthy control cynomolgus monkeys  encoding 373 amino acids was tagged with flag at the N terminus and was inserted into a lentiviral vector for monkey-fertilized egg injection. The lentiviral vector contains the mouse prion promoter,35 In brief, adult females were hormone stimulated, and their oocytes were recovered for in vitro fertilization and culture. Metaphase-II arrested oocytes were selected for perivitelline space injection of lentiviral solution. After virus injection, the oocytes were fertilized by intracytoplasmic sperm injection, followed by in vitro culture. Embryos at the 4–8 cell stage were selected for embryo transfer based on morphological appearance. Surrogate females at synchronized reproductive cycles were identified based on their menstrual cycle. Pregnancy was confirmed by B-ultrasound 30–35 post-transplantation days. The surrogates delivered newborn monkeys naturally after around 160 days of gestation.Assisted reproductive techniques in cynomolgus monkeys have been described in our previous study.49 In short, after monkeys fasted for at least 8 h, a lumbar puncture was performed at the intervertebral space L3-L4 or L4-L5 using a standard needle. CSF was collected into a 1.5 ml sterile microtube  and immediately frozen at −80 °C. Blood samples were obtained on the same day as the lumbar puncture in fasting conditions. Whole blood was drawn with a 20 G needle gauge into a 5 ml EDTA tube. Tubes were gently inverted 5–10 times and centrifuged at ×3000 g for 10 min at 4 °C. The supernatant was aliquoted in volumes of 200 μl into EP tubes and immediately frozen at −80 °C. The samples were processed at room temperature. The time between collection and freezing of both CSF and plasma sample was <30 min. All CSF and plasma biomarkers including total tau, p-tau231, NFL, Aβ40, and Aβ42 were analyzed by Simoa platform of G-Bio Co., LTD, Hangzhou, China.The CSF sample collection and assay followed standard protocols and have been described previously.g for 10 min. The supernatants were loaded to SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% skim milk/PBS for 1 h at room temperature. Primary antibodies were diluted in 3% BSA/TBST  and incubated with the blot membrane overnight at 4 °C. The blotted membrane was washed in TBST 3 times (each time 5 min), followed by incubation with HRP-conjugated secondary antibodies in 5% skim milk/TBST for 1 h at room temperature. After three washes in TBST, ECL plus  was used to detect immunoreactive signals on the membrane.Primary antibodies to Flag , NeuN  GFAP , IBA1, β-Amyloid,1–16 , β-Amyloid, 17–24 , β-Amyloid 1–42, Amyloid Precursor , Amyloid Precursor , APP C-Terminal fragment , GAPDH , Neurofibrillary tangles , Tau , Phospho-Tau , β- Amyloid 1–42 , sAPPβ, and Oligomer  were used. For western blotting, Tau1 and WT tissues were lysed in ice-cold RIPA buffer  containing Halt Protease Inhibitor cocktail (Thermo Scientific) and PMSF. The tissue lysates were incubated on ice for 30 min, centrifuged at 12,000 × 50 In brief, freshly isolated monkey brain tissue blocks were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde for 48 h and sectioned using a vibratome. All sections used for electron microscopy were dehydrated in ascending concentrations of ethanol and propylene oxide/Eponate 12 (1:1) and embedded in Eponate 12 . Ultrathin sections (60 nm) were cut using a Leica Ultracut S ultramicrotome. Thin sections were counterstained with 5% aqueous uranyl acetate for 5 min followed by Reynolds lead citrate for 5 min and examined using a Hitachi  H-7500 electron microscope.The procedure of EM examination has been described in our previous studies.t test for comparing two groups when the data follows a normal distribution and the variances of the two groups are assumed to be equal. When comparing groups with small sample sizes or when the data are skewed or have outliers, the Mann-Whitney test was employed. When the same subjects were measured under different conditions or at different time points, we used repeated measures t-test. For three or more groups, one-way ANOVA with Tukey’s multiple comparisons tests was used. Calculations were performed with GraphPad Prism software. The quantitative results are presented as mean ± SEM. P < 0.05 is considered a significant difference.Statistical significance was assessed using the 2-tailed Student’s Supplemental materialsShort-term memory test of WT and Tau3 monkeys at 38 months of ageLong-term memory test of WT and Tau5 monkeys at 40 months of ageSleep monitoring of WT and Tau2 monkey at 38 months of ageFine finger coordination assay of WT and Tau3 monkey at 30 months of ageThe body movements of Tau1 monkeys at 25 and 35 months of age


This article has been corrected: The authors corrected Table 3, “Sequence of primers for RT-PCR and long PCR,” because they forgot to update this table before submission. The primers used in the study were changed three times before a set that worked was synthesized and verified by “Sangon Biotech.” They also found and corrected a duplication in Figure 5B created during the figure assembly - column chart “PGC-1α” replicates column chart “Fis1.” Correction was done with data from the original sets of Western blots for PGC-1α protein. These corrections have no impact on the experimental outcome or conclusions.Table 3 and Figure 5 are presented below.The corrected


Widely tunable coherent sources are desirable in nanophotonics for a multitude of applications ranging from communications to sensing. The mid-infrared spectral region (wavelengths beyond 2 μm) is particularly important for applications relying on molecular spectroscopy. Among tunable sources, optical parametric oscillators typically offer some of the broadest tuning ranges; however, their implementations in nanophotonics have been limited to narrow tuning ranges in the infrared or to visible wavelengths. Here, we surpass these limits in dispersion-engineered periodically poled lithium niobate nanophotonics and demonstrate ultrawidely tunable optical parametric oscillators. Using 100 ns pulses near 1 μm, we generate output wavelengths tunable from 1.53 μm to 3.25 μm in a single chip with output powers as high as tens of milliwatts. Our results represent the first octave-spanning tunable source in nanophotonics extending into the mid-infrared, which can be useful for numerous integrated photonic applications. A photonic chip synthesizes light over an octave bandwidth reaching into the mid-infrared. Widely tunable coherent sources are vital for applications ranging from multichannel optical communications (Previous efforts toward OPO miniaturization include using lithium niobate–diffused waveguides with fiber feedback loops ((3)] and quadratic [χ(2)] nonlinearities  and momentum (pk = sk + ik + 2π/ΛQPM), so they can be tailored by engineering the waveguide dispersion (s/∂ωp) is given by the ratio of group velocity differences (1/iv − 1/pv)/(1/iv − 1/sv), while the gain bandwidth is inversely proportional to 1/iv − 1/sv. We have engineered the dispersion of the poled waveguide to balance these effects. As a result, a small change in the pump wavelength produces large changes in the output wavelengths while maintaining a predictable tuning curve without substantial mode competition (see fig. S1).The simulated tuning behavior of four OPOs with different poling periods is shown in To study the transient and steady-state behaviors of the nanophotonic OPOs, we use pulses that are much longer than the cavity lifetime of the OPOs. This arrangement also allows us to use low average powers incident on the chip while maintaining high peak powers. The experimental setup is shown in The measured OPO on-chip signal power at ~1950 nm is shown in 2 buffer layer exhibits an absorption peak . For our device, this ratio is ~9% at 1950 nm, indicating that the output coupling is small compared to the total losses in the resonator. This could be caused in part by the little transmission of the output coupler, particularly at mid-infrared wavelengths, and in part by intrinsic resonator losses and losses at the input coupler. Fine tuning of the coupler designs and reducing the cavity loss can lead to substantial improvement of efficiency. We used adiabatic couplers in this work since they provide a simple means to approximately achieve our requirements of high signal and idler coupling together with low pump coupling. However, the input coupler should, ideally, have 100% coupling at signal and idler frequencies since any transmission in this coupler behaves as additional resonator loss, leading to higher thresholds and lower efficiencies.Simultaneously, the input coupler should provide very low coupling at the pump wavelength, since any coupling just leaks pump power into the unused port, and also provides an undesired feedback path for the pump. These characteristics may be achievable through more advanced coupler designs, for instance, those obtained by inverse design methods . The input and output couplers are identical and are designed so that signal and idler wavelengths (λ > 1.8 μm) have large coupling factors (>80%), while pump wavelengths near 1 μm are only slightly coupled (<10%). The residual coupling of the pump leads to round-trip feedback factors of less than 1% that produce negligible modulations of the pump intensity as a function of frequency, allowing continuous tuning of the pump wavelength.When designing a tunable OPO, it is desirable to have a large tuning slope so a small change in pump wavelength produces large changes in the output wavelengths. At the same time, a small gain bandwidth is preferable to limit the number of resonator modes experiencing gain. To achieve a balance between these two behaviors, we engineer the dispersion of the waveguide using its geometry, resulting in 2.5-μm-wide waveguides on a 700-nm-thick lithium niobate layer and 250 nm of etching depth. The mode profile for a set of representative wavelengths is shown in fig. S1B, illustrating that the modal overlap remains substantial despite the large frequency difference.2 buffer layer. We provide quasi-phase matching in a 5-mm-long region through periodic poling  to 2.5-μm-wide waveguides inside the resonator. To maximize the spectral range covered on a single chip, we fabricated OPOs with poling periods ranging from 5.55 to 5.7 μm in 10-nm steps. We include a straight waveguide next to each OPO for calibration and quasi-phase matching verification  with an x-cut, 700-nm-thick MgO-doped lithium niobate layer and a SiOWe characterize our OPOs using the experimental setup shown in 5, which translate to losses below 0.3 dB/cm for waveguides without poling. Detailed inspection of the periodically poled waveguide inside the resonator reveals periodic roughness of the waveguide sidewalls, likely from the polarization-dependent etch rate of lithium niobate. More studies are needed to improve the resonator quality factor.To estimate propagation losses in our waveguides, we fabricated chips with arrays of critically coupled resonators and extracted quality factors ~6 × 10To estimate input and output coupling losses, we use single-mode lensed fibers to couple into and out of the chip and then divide the total throughput loss equally between both interfaces. We do this on several unpoled straight waveguides and obtain a coupling coefficient varying from 10 to 13 dB, with 10 dB giving the most conservative estimate for on-chip input power. When using an asymmetric setup , we assume that the input coupling remains at 10 dB and calculate the output coupling from the throughput loss. Comparing transmission of straight waveguides to that of OPOs allows us to estimate a total loss factor of 0.929 per coupler at the pump wavelength, reasonably close to the simulated value of 0.95 (fig. S1A). The plots of 0ηe. The internal efficiency (η0) measures how efficiently pump photons are converted into signal and idler photons, while the escape efficiency (ηe) measures the fraction of the generated signal and idler photons available at the output of the OPO. The difference between the pump power at the beginning and end of the gain section is ∆pP = pP(0) − pP(gL). The internal efficiency is just the pump depletion η0 = ∆pP/pP(0) shown in The efficiency of an OPO (η) can be written as the product of two efficiencies, η = ηe = (ω/ωp) T(ω)/[1 − L(ω)], where T(ω) is the power transmission coefficient of the output coupler, while L(ω) is the total round-trip power loss factor of the resonator, i.e., L(ω) = [1 − T(ω)]2 exp(−2αL), where α is the field propagation loss, assumed constant, and the square term comes from considering identical input and output couplers. Since the output power can be calculated from the efficiency as Pout(ω) = η0ηe(ω)pP(0), the idler power can be estimated from the signal power and the simulated coupler response, T(ω), asThe escape efficiency is given by η


The number Ns of entering flux quanta in different triangular voids of the SG is proportional to the linear size s of the void, while the field periodicity of flux jumps varies as 1/s. We explain this behavior by modeling the triangular voids in the SG with effective superconducting rings and by calculating their response following the London analysis of persistent currents, Js, induced by the applied field Ha and by the entering flux. With changing Ha, Js reaches a critical value in the vertex joints that connect the triangular superconducting patches and allows the giant flux jumps into the SG voids through phase slips or multiple Abrikosov vortex transfer across the vertices. The unique flux behavior in superconducting SG patterns, may be used to design tunable low-loss resonators with multi-line high-frequency spectrum for microwave technologies.We study the magnetic field response of millimeter scale fractal Sierpinski gaskets (SG) assembled of superconducting equilateral triangular patches. Directly imaged quantitative induction maps reveal hierarchical periodic filling of enclosed void areas with multiquanta magnetic flux, which jumps inside the voids in repeating bundles of individual flux quanta Φ They are frequently reported in materials studies (from molecular assemblies3 to domain structures in quantum magnets4), and are often employed in technological devices (from compact antenna designs5 to efficient heat exchangers6 and advanced load supports7).Fractal structures with self-similar repetition of topologically identical features at diminishing length scales are universally found in nature  with different scale array of voids. Prior studies of SGs comprised of SC wires or wires with Josephson Junctions which showed distinct hierarchical and repetitive changes in resistivity and inductance of the samples in applied fields near the SC transition temperature (Tc)15. These samples were lattices of Sierpinski gaskets up to 6th order with elementary triangles of submicron or a few micron size. In small applied magnetic fields, it was possible to successively fill different triangular subsets composing the SG with individual magnetic flux quanta, Φ0 = πħ/e. The hierarchy of flux filling, resulting in sharp changes of Tc or inductance of the SG arrays, followed digital flux quantization rules, NΦ0 → (N ± 1)Φ0, commonly reported for multiply-connected superconductors, with specifics imposed by the fractal pattern geometry. For experiments close to Tc, the data analysis is simplified due to negligible Meissner screening, resulting in homogeneous magnetic field distribution (see16 and refs. there). However, at low temperatures (T), where losses are desirably minimized, the screening effects become important and the magnetic field is modified by SC persistent currents. Moreover, due to increased critical currents at low T, the flux entry into the samples is strongly delayed and may depend on the dynamics of phase slips or entry of Abrikosov vortices which can transfer single or multiple flux quanta into the voids inside the superconductor.In particular, Sierpinski gaskets (SG), formed by triangles of progressively decreasing size  transition temperature Tc = 8.75 K, grown by high-vacuum magnetron sputtering. A Sierpinski gasket obtained after successive removal of progressively decreasing triangular areas while leaving narrow 1 µm bridges between vertices of the remaining triangular SC patches is shown in Fig. order gasket is a simple equilateral triangle and the order increases upon successive addition of wires connecting the centers of the larger triangle sides. In our case the 0-order gasket corresponds to three triangular patches surrounding the central triangular void. The equivalence with the 0-order wire gasket is that we similarly begin with one hole in the SC structure. Below we present the main results for our highest 3d-order SG pattern c were observed using MOI. The samples were mounted on a cold finger of a commercial Montana cryostat and covered by an indicator film with a large Verdet constant to spatially visualize the normal magnetic field at the sample surface, Bz, in polarized light. Careful calibration of image intensity versus applied normal field Hza at T slightly above Tc allows accurate quantitative assessment of induction distributions in the sample.The macroscopic magnetic response of the samples in a magnetic field was measured using SQUID magnetometry, and the flux distributions at T < Td-order SG structure using a set of MOI pictures obtained by gradually increasing the applied field, Hza, where the image intensity corresponds to the local strength of Bz. Quantitative changes of local Bz within various triangles in our SG samples will be presented below as Bz(Hza) plots.We start with demonstration of the magnetic flux entry in our 3z map in the sample with increasing Hza in steps of ΔHz ~ 0.03 Oe at T = 3.5 K. At this temperature, the magnetic field ≲ 0.4 Oe is mostly screened from the entire sample by Meissner currents JM . Specifically, small negative Bz↓↑Hza (dark contrast as opposed to bright Bz↑↑Hza) is observed at the sides and in the vertices of the TVs located adjacent to the outer-edges of the SG sample as marked by longer arrows in Fig. Figure  JM Fig. b. Bz incz (bright contrast), emanates from the vertices of the smallest voids, TV4, at the sides of larger internal TVs as marked by short arrows in Fig. M distributed over triangular patches of our SG. In the Meissner state, in a multiply connected SC structure with a single hole, such as a ring, the screening current, JM, is concentrated near the inner and outer ring edges but has the same polarity across the ring’s width21. As a result, the applied field is enhanced at the outer ring edge, while the local negative (opposite to Hza) field appears at the inner edge .With slow increase of H2 (s = 0.5 mm) marked as 2–3–4 in Fig. 2-#2 and -#3 are filled simultaneously and TV2-#4 is filled at slightly larger Hza. The abrupt flux jumps are accompanied by the dark-to-bright reversal of contrast at the TV2 edges, as described above for TV1. Following the jump, the field in TV2 is higher than Bz in TV1 but the flux change is smaller (ΔΦ2 ~ 2600 Φ0) due to the smaller triangle area.With further increasing field, the jump-wise flux filling occurs in the next smaller sized, TV2, the next smaller TV3 voids  begin to fill with magnetic flux at Hza > 0.8 Oe  begin filling  = Bz(Hza + 0.03 Oe)-Bz(Hza) corresponding to appropriate changes of the currents ΔJ  during the jump. Figure i in any sized TVi yield qualitatively the same picture of a homogeneous ΔBz over the main TVi area with enhanced positive ΔBz at the TVi periphery. Pronounced contrasting features emerge in three neighboring triangular regions around the TVi. They have the same size as TVi, but contain smaller triangular voids surrounded by SC patches. Together with the central TVi where the flux jump occurred (brightest contrast), these neighbors make up the Sierpinski sub-gaskets (sub-SG) of a lower order. Such smaller sized sub-SGs of order 2, 1 and 0 are isolated with dashes in Fig. Important details of the changing current patterns during the flux jumps in our samples are revealed by difference images presented in Fig. z patterns show that after the flux jumps into a TVi, the screening currents JM in the SC patches surrounding the TVi are inverted along the edges of the TVi and also along the sides of the smaller TVs making up the sub-SGi structure. At the same time, JM is noticeably reduced at the sub-SGi’s outer boundary. The picture corresponds to the changes from pattern (a) to (b) in current distributions sketched in Fig. za beyond the jump field, the sub-SGi Bz-maps (not shown) restore the pre-jump features qualitatively similar to Fig. The ΔBza, at larger fields we observe unexpected local negative flux jumps, as illustrated by dark triangles (ΔBz < 0) in Fig. z in prior flux filled TVs is accompanied by a partial positive ΔBzp in their neighbors  averaged over the area of individual TVs, and transformed IMOI into a median MOI(Bz) calibration. Multiplying the obtained i acquired by the TVi. Figure zap) plots for TVs of all four sizes composing the SG. The is are periodic. They have basically the same amplitude and are separated by identical field gaps ΔHza between jumps. The height of the jumps Δ i size.To quantitatively analyze the magnetic flux evolution in our SG pattern, we measured the MOI signal  are ~ 650:1350:2650:6600 (~ 1:2.1:4.1:10.2), i.e. ΔΦi changes practically linearly with s (logΔΦ-logs fit gives ΔΦ ~ s1.131). Assuming that the SC currents, screening the applied field or Bz in the TVi due to flux jumps, are concentrated along the sides of the SC triangular patches and at their vertex links, we can model the individual sub-SGs as narrow rings with effective radius R = (rinRci)1/2 = s/61/2, intermediate between the inscribed (rin) and circumscribed (Rci) circles confining the TVi. The ring width was chosen as w = 1 µm, corresponding to the width of the bridges between all triangular patches. Appropriate values of inductance L of four of our sub-SGs calculated using formula for narrow rings18, L = µ0R[ln(8R/w) − 2 + ln4], shown by squares in Fig. i (round dots) in TVs of different size. This indicates that the inductance of the sub-SGs defines the size of the flux jumps in their central voids.The distribution of successive flux jump amplitudes ΔΦ13 or similar samples containing Josephson junctions in the wires15. Macroscopic transport and susceptibility measurements on these samples revealed a rich hierarchy of sharp changes of the transition temperature, Tc(Hza), and inductance, L(Hza), corresponding to the complex filling of different size triangles composing the SG with single flux quanta. The theoretical treatment of these results was usually based on the Ginsburg-Landau (GL) equations25 assuming the homogeneous magnetic field distribution, i.e. neglecting the SC screening fields. Basically, the superconducting nature of the samples was accounted through the field dependent phase relations of the SC order parameter, which dictate the flux quantization in multiply connected samples. In the case of SG, the flux quanta are predicted to enter the n-order SG with elementary (minimum size) triangles of area A0 at fields H > Hc = Φ0/(4nA0)16. In our SG formed by SC patches, A0 is the area of the smallest triangular void, yielding Hc ~ (1/4n)3 × 10–3 Oe, which is much smaller than the observed flux entry fields , while the values of flux jumps we measure are much larger than Φ0. At the same time, theoretical expectation for successive flux entry, starting from the largest triangle and proceeding to smaller triangles with increasing Hza, is consistent with our observations . However, in our case, the succession of flux entry in different sub-SGs is defined by a distinct mechanism which we discuss below.Experiments on superconducting Sierpinski samples were previously realized on periodic lattices of different order SGs with basic triangles of narrow few-micron long SC nanowiresc/2 the screening effects are important. Under these conditions, the flux penetration into TVs should occur either through phase slips or by the transit of Abrikosov vortices across the 1 µm bridges connecting the triangular SC patches. Flux penetration occurs when the screening current in these bridges acquires a critical value Ic. The screening currents flowing over the patches converge in the narrow bridges yielding there the enhanced current density, and with increasing Hza, the total current reaches Ic first in these regions. The resulting phase slips or moving vortices temporary suppress the SC order parameter |Ψ| near the vertices of the central TVis in the sub-SGs and provide channels for flux entry. Clearly, the largest current is initially achieved  equations39. These works explained many experimental observations of sharp changes in the microscopic SC ring properties due to the periodic entry of single flux quantum Φ0, such as oscillations in Tc, resistivity, susceptibility, inductance, and heat capacitance44.To understand the regularity and large values of the flux jumps in different TV33 accounting for different relaxion times of the phase (τφ) and amplitude (τ|Ψ|) of the SC order parameter in relatively large rings (R ≳ 10ξ) showed that transitions between many metastable states with different vorticity Lv can yield ΔLv >  > 1 . These transitions repeat at appropriately large field steps (ΔH). They occur if τ|Ψ|> > τφ through phase slips with complicated temporal and spatial variation of φ and |Ψ| depending on the values of relaxation parameters, radius and width of the ring, and ξ, when the gauge-invariant momentum of the SC pairs reaches a critical value pc 36.However, computer simulations of TDGL equationsmax = 11 at Happ < 40 Oe and gradually decreasing ΔL at larger fields were found in narrow 4µm Al ring at T < Tc/345. ΔL = 3 jumps were reported for 2µm Al rings33. Later, giant flux jump transitions between metastable SC states with ΔLv up-to 70 were detected through sharp changes of the low-T tunnel current in narrow 25 µm square Al rings with a normal electrode in one corner47.In earlier experiments, giant flux jumps with ΔL49. Unlike the GL formalism, which is mostly applied to mesoscopic rings, the London description is based on electrodynamics equations appropriate for any sample size, while anchoring the flux quantization requirement that maintains the coherent state in the SC material of the ring.The most intuitive and clear picture of flux quantization in multiply-connected SC samples appears in the London description of the induction and current pattern variations in rings2/d for ring thickness d << λ), where one can neglect the field induced by the screening currents, the periodic flux entry was explicitly described in49. Transitions between states with N and N ± 1 flux quanta in the ring were suggested to occur via the nucleation of Abrikosov (or Pearl for d << λ) vortex or antivortex, either at the outer or inner ring edge, and its motion across the ring width, thus adding or removing one Φ0 in the ring annulus. The barrier for this process is defined by the vortex nucleation field. Interestingly, at some fields H ~ (N1 + N2)/2 the energy for N1 and N2 states with |N1 − N2|> > 1 is the same, which could in principle allow large changes of vorticity in the ring.For thin SC rings with dimensions much smaller than the Pearl length  jumps inside the annulus, the screening current at the inner ring edge changes direction to support Φ, and with further increasing field, the Meissner screening current pattern restores itself until the next flux entry. This picture corresponds to changes of the MOI patterns observed around different voids in our SG samples.For large SC rings, where the self-induction contribution becomes important, and where the flux jumps with large vorticity were predicted by the GL calculations, the accurate description of the magnetic response accounting for the self-field induced by the Meissner current was given in20 Brandt and Clem calculated the SC ring energy in a homogeneous applied field Ha = Ba/µ0, accounting for the screening currents j in the presence of a fluxoid inside the ring and the fields Bj induced by these currents:InB = Ba + Bj and the second term is the kinetic energy of the currents. The total current and vector potential were divided into parts driven by the fluxoid and by the applied field respectively . Finally, the Gibbs potential G = E − mBa/2 was obtained, which describes the state of the ring accounting for the Ba source inducing the magnetic moment m in the ring. Depending on the applied field, minima of G defined stable flux values in the ring annulus with neighboring states distinct by ± 1Φ0.Here, the first term is the energy of the total field i containing central triangular void TVi with side length s as narrow ring with effective radius R = (rinRci)1/2 = s/61/2 and follow the same calculations as in20 (see Supporting Info i (omitting the constant homogeneous applied field contribution):If we approximate the sub-SGeff = πR2 = (π/6)s2 is the effective area of the TVi in the sub-SGi and C = [tanh−1(a/b) − 1 + ln4] comes from the inductance of a narrow ring of width w, inner radius a = R − w/2, and outer radius b = R + w/2. The minima of GN correspond to the multiquanta states defined by Aeff and Ba. However, transitions between different states are delayed until Ba reaches a characteristic value allowing either phase slips or nucleation and transit of Abrikosov (Pearl) vortices across the narrow bridges in the corners of the TVi. These fields are reached when the total screening current in the bridge acquires a critical value Ic, which yields the flux jump in the TVi: Φ = NΦ0 = Ls-SGIc (Ls-SG is the inductance of the sub-SG). After the flux jump, the total current in the bridge vanishes (− jΦ screening the fluxoid inside TVi and + jH screening the applied field compensate each other). With further increasing Ba, jH restores the Meissner distribution over the entire bridge until the total current reaches Ic again and an additional fluxoid Φ = NΦ0 jumps in. In small fields, as in our experiment, which do not affect the critical current, the jumps should be periodic in field, repeating in steps of ΔBa = Ls-SGIc/Aeff.Here Ais of all our sub-SGs, should have the same Ic. However, due to the hierarchical current flow in the entire sample, the critical current is first achieved near the vertices of the largest TV1. After the flux enters the largest TV1 and the total current through it’s bridges vanishes (∫(jH-jΦ)dr = 0), the current trajectories form closed loops in the three neighboring smaller sub-SGs and reach Ic at their respective TVi bridges with further increasing Ha, resulting in subsequent flux jumps in these TVis. Similar scenario repeats for the next smaller sub-SGs. The flux jumps for smaller structures occur between repeating jumps in larger sub-SGs.Similar 1 µm bridges in TV51 or due to the vortex transfer48 across the narrow bridge at the vertices. However, quantitative estimates show a faint probability of phase slips in our samples: P ~ exp(− ΔF/kBT) with the barrier height ΔF ~ 104 kBTc and appropriate critical current density Jc ~ 2MA/cm2  vortex transfer to fill the TVi with multiple flux quanta, NΦ0. Considering different sub-SGis, independently, the fluxoid vorticity N is proportional to the inductance Ls-SG of the sub-SGi, which can be approximated by a narrow ring of the order of sub-SGi TVi size s, so that Ns-SG ~ s. In turn, the field periodicity of flux jumps ΔHa ~ 1/s.The succession of flux jumps into central triangular voids, TVi reverses its direction. The flipped current may compensate the Meissner current induced by Ha and the total SC current in the TVi joints vanishes, allowing larger current collection at the narrow bridges of smaller TVs and enabling their flux filling. Eventually, multiquanta flux jumps repeat, alternating between large and small sub-SGs where appropriate Ns-SG enter at appropriate Ha.We observe changes of the current patterns during the flux jumps when the screening current around the TVWe anticipate that the superconducting Sierpinski structures, where regular giant flux jumps are induced by small applied magnetic fields, may be used for designing low-loss tunable resonators for information and communication technologies. Fine changes in the inductance of the SG pattern due to the controlled fast flux entry in separate sub-SG can allow controlled switching in high frequency operations, in/out signal delivery, and exchange between elements of quantum electronics devices . The characteristic zero-field frequency response can be adjusted by the SG size and form a wide band of resonance lines depending on the SG order.The samples were fabricated by lift-off procedure of 100 nm niobium film deposited with high vacuum DC magnetron sputtering on a photoresist pattern prepared using laser lithography on a silicon wafer. The accuracy of all 1 µm bridges between triangular patches forming the resulting niobium SGs was inspected in an optical microscope using 100 × objective.z on their surface in a polarized light microscope. To improve the signal/noise ratio, the magneto-optical images were accumulated using multiple-exposures in a digital 16 bit camera with cooled 1024 × 1024 CCD array. The image intensities I were transformed into the Bz-maps using accurate B-I calibration obtained slightly above the superconducting Tc. Digital operations with images were performed using image processing software.The silicon chips with niobium SG structures were mounted on the cold finger of specially designed optical castle in a helium closed cycle Montana cryostat. A magneto-optical indicator with large Verdet constant was placed on top of the samples, allowing images of the normal magnetic field distributions BThe description of TDGL simulations of the current distributions in the large Sierpinski gasket without and with magnetic fluxoid in the central triangular void are presented in the Supporting Info, where we also show details of our London calculations of the sub-SG Gibbs potential defining the giant flux jumps in our samples.Supplementary Information.


Cobalt oxide, nickel oxide and cobalt/nickel binary oxides were synthesised by electrodeposition. To fine tune composition of CoNi alloys, growth parameters including voltage, electrolyte pH/concentration and deposition time were varied. These produced nanomaterials were used as binder free electrodes in supercapacitor cells and tested using three electrode setup in 2 MKOH aqueous electrolyte. Cyclic voltammetry and galvanostatic charge/discharge were used at different scan rates (5–100 mV/s) and current densities (1–10 A/g) respectively to investigate the capacitive behaviour and measure the capacitance of active material. Electrochemical impedance spectroscopy was used to analyse the resistive/conductive behaviours of these electrodes in frequency range of 100 kHz to 0.01 Hz at applied voltage of 10 mV. Binary oxide electrode displayed superior electrochemical performance with the specific capacitance of 176 F/g at current density of 1 A/g. This hybrid electrode also displayed capacitance retention of over 83% after 5000 charge/discharge cycles. Cell displayed low solution resistance of 0.35 Ω along with good conductivity. The proposed facile approach to synthesise binder free blended metal electrodes can result in enhanced redox activity of pseudocapacitive materials. Consequently, fine tuning of these materials by controlling the cobalt and nickel contents can assist in broadening their applications in electrochemical energy storage in general and in supercapacitors in particular. However, these renewable and sustainable sources of energy are intermittent in nature and require appropriate energy storage systems. Energy storage devices such as rechargeable batteries and supercapacitors (SCs) are becoming extremely widespread to address the intermittency of these renewable and sustainable sources of energy. SCs have attracted immense research interest recently since these devices can bridge the energy and power densities gap between rechargeable batteries and traditional electrostatic capacitors2. Furthermore, characteristics such as exceptionally high-power densities, outstanding stabilities and excellent efficiencies make SCs highly desirable electrical energy storage devices. Based on the charge storage mechanism, SCs can be divided into two main categories i.e., electric double layer capacitors (EDLCs) and pseudocapacitors (PCs). In EDLCs, charge is stored on the electrode/electrolyte interface through physical ionic adsorption. Capacitance in EDLCs depends on the active material’s accessible surface area and the distance between centres of electrolyte ions and atoms of the active material. Carbon based materials such as activated carbon, graphene, carbon nanotubes and polymeric carbon are the most commonly used electrode materials in EDLCs4. Whereas pseudo-capacitive energy storage is based on fast and fully reversible Faradic reaction involving electronic transfer at or near the surface of electrodes6. Typical electrodes of PCs are based on transition metal oxides, hydro oxides or conducting polymers. Electrode is a key component of a supercapacitor cell since it can have a significant impact on its performance. Therefore, selection of suitable electrode material is of utmost importance. The advantages in using metal oxides based nanostructured materials for supercapacitor applications are generally attributed to their large surface area7, exceptional mechanical, and electrical properties by virtue of their confined dimensions, short diffusion path lengths and high theoretical capacitance9. Different nano-structures with particular architectures including nanorods, nanowires, nanosheets, nano-cubes and nanospheres have been intensively utilised and widely reported in literature as advanced electrode active materials for SCs10.Contribution of energy generated through renewable and sustainable sources such a wind, tidal and solar is growing significantly due to the repaid growth in demand and impact of fossil fuel-based energy production on the environment2 is considered the most promising electrode material when compared with other transition metal oxides since it has superior theoretical capacitance and excellent electrical conductivity. However, toxicity and elevated cost associated with RuO2 makes it less desirable for its application as an efficient active material for SCs. Consequently, wider commercialisation and real word applications of SCs require nanomaterials based on traditional metal oxides/hydro oxides which are inexpensive, and less contaminating. Different transition metal oxides such as MnO2, NiO and Co3O4 have been investigated intensively with an aim to deliver superior capacitive performance in a cost effective and environmentally friendly manner. Nickel and cobalt oxides are of enormous interest as pseudocapacitive electrodes due to their high specific capacitance, energy densities, thermal/chemical stabilities, ease of fabrication, as well as low-cost and environmental benignity12. NiO, like Co3O4 has been widely evaluated as an electrode material in SCs due to its cost-effectiveness, natural abundance, and good specific capacitance. NiO is one of the most research metal oxide for SC’s applications, however, inferior electrical conductivity and poor reversibility are some of the key disadvantages associated with the wider adoption of NiO. Recent literature suggests that transition metal oxide based composite materials exhibit better performance than a single or individual material for supercapacitor applications thanks to their enhanced chemical activity, stability and electrical conductivity13. Therefore, coupling NiO with other materials such graphene, activated carbon and metals oxides such as Co3O4 has resulted in improved capacitive performance where each component complements other and actively contribute towards overall performance enhancement18. Surface morphologies and deposition methods can also have a considerable impact on the electrochemical performance of SC cell since these can greatly impact charging and discharging processes19. Furthermore, use of organic binders during the production of electrodes for SC cells can have a negative impact on the performance of these device, as binders are usually comprised of insulating materials which can result in increased material resistance due to immobility in binder and stocking effect in electrode materials20. While ‘‘binder free electrodes’’ are a novel approach to overcome the limitation posed by these traditional binders. With the exclusion of binders, researchers are seeking to enhance the performance of electrochemical energy storage devices i.e., supercapacitors while making these devices more environmentally friendly. Binder free electrodes are typically fabricated through the direct growth of active material on current collector’s surfaces or by using self-standing films. Electrolysis is a promising method to deposit metal nanostructures directly onto current collector’s surfaces such as carbon cloth (CC), followed by directly using these as binder-free electrodes for SC’s applications. Selection of CC as current collector in this particular study is due to added benefits such as the 3D porous network structure of CC to adsorb more ionic electrolyte together with the outstanding electrical conductivity21. The establishment of electrolytic cell with highly conductive CC as working electrode, the nanostructures can be directly deposited on CC easily by electrolysis. The deposition without organic additive is advantageous to bring the best of the synergetic effects of metal and highly conductive carbon. This study distinguishes itself from other works on three key merits which are successfully incorporated in a single study, (1) effective synthesis of Co, Ni monometallic oxides and CoNi binary oxides with precisely tuned composition using facile electrodeposition method, (2) production of binder free transition metal oxides-based electrodes through electrodeposition eliminating the requirement of non-conducting binders, (3) manufacturing of very thin and light weight electrodes where lightweight/thinness of electrodes can be highly beneficial for the superior capacitance of a SC cell. Since, electrode material only on/near surface actively contributes towards electrochemical performance of a SC whereas light weight electrodes can have superior capacitance performance as given by Eq. , the counter electrode was kept at 3 cm from CC. All reagents used were of analytical grade and used without any chemical alteration. The precursor for the deposition of cobalt consisted of 0.2 M CoSO4⋅7H2O and 0.5 M of H3BO3. pH was adjusted to 3.3 by adding dil. H2SO4. The voltage of deposition was 4 V. Nickel nanostructures were deposited from a solution of 0.2 M NiSO4⋅6H2O and 0.5 M of H3BO3, with pH 3.8 under constant voltage of 4 V. Nanocrystalline Co–Ni alloys were electrodeposited on CC from an electrolyte solution which consisted of CoSO4⋅7H2O, NiSO4⋅6H2O and H3BO3. Growth parameters including voltage, electrolyte pH, duration of deposition, electrolyte concentration was changed in order to alter alloys composition. The optimum electrochemical characteristics were obtained for Co–Ni alloy prepared with a deposition voltage of 3.5 V, and deposition duration of 1 h from an electrolyte solution contained 0.1 M of CoSO4⋅7H2O, 0.1 M of NiSO4⋅6H2O and 0.1 M H3BO3. The pH of the solution was 3.61.To obtain nanostructure coated carbon cloths, the metal nanostructures were deposited within the channels of the carbon cloth following the optimized fabrication conditions and protocolsThe metals and their alloys deposited on CC were subjected to X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDX), and Elemental Mapping. The morphology and microstructure were observed by SEM (FESEM thermoscientific Apreo C), XRD (Bruker D8 ADVANCE diffractometer) with CuKα radiation and by X-ray Photoelectron Spectroscopy ). The chemical composition of the nanostructures was analyzed using EDX and elemental mapping .The X-ray crystallographic analysis was used to study the structural analysis of Ni and Co and CoNi alloy as shown in Fig. 26. The elemental compositions from EDX were found to be 81.6 (wt%) of Co and 18.4 wt% of Ni. If the layer of the deposited film is thick enough, the X-rays beam cannot penetrate all the way through the film which weakens the signals from the underlying material. That explains a weaker CC peak in Co sample.The XRD pattern of the deposited Co–Ni alloy is shown in Fig. The typical morphology of the deposited nanostructure in CC is shown in Fig. The more detailed elemental composition and electron structure of the as-prepared CoNi hybrid deposited on CC was further analysed by XPS where the working pressure in the spectrometer chamber was ~ 10–9 mbar. Flood gun was used for charge compensation and spot size was 400 µm spectra, broad XPS spectra is displayed in Fig. 1/2 and 2p3/2 resulting from the spin–orbit splitting of Ni2p and Co2p is shown in Fig. 3+25, with the satellite peaks at 863.37 eV and 880.06 eV. The Co 2p3/2 and Co 2p1/2 peak at 782.09 and 797.12 eV shown in Fig. 2+ whereas the peaks at 785.4 eV and 798 eV are ascribed to satellite peaks. The typical metal–oxygen bonds are at the O1 peak at 529.1 eV26 and the peak at 531.2 eV corresponds to OH group27. The peak of metal–oxygen can’t be observed in the current scan and the O1 peak located at 531.9 eV can be assigned to OH group. The peak at 533.1 eV might be due to defects, contaminants, or multiplicity of physic-/chemisorbed water at/within the interface of the material30.Figure 2), the platinum wire was used as a counter electrode, Hg/HgO was used as a reference electrode and 2 MKOH aqueous solution as an electrolyte. The electrochemical behaviour and capacitance were evaluated using cyclic voltammetry (CV) and galvanostatic charge–discharge (GCD) measurements, recorded at different scan rates and current densities respectively. The electrochemical impedance spectroscopy (EIS) analysis was performed at frequency ranges from 100 kHz to 0.01 Hz at applied voltage of 10 mV to understanding the conductive/resistive behaviour of active samples. The specific capacitance (Qs) was calculated from the discharge curve using the following formulae:The electrochemical performance of active material was assessed using the Biologic VSP-200 in a typical three-electrode setup. For the three-electrode configuration, prepared materials deposited on CC were directly used as working electrodes (1 cmwhere I (A) represents the discharge current density, ∆t (s) represents the discharge time, m (g) is the mass of active material, ∆V is the voltage window.Specific energy , and specific power  of the supercapacitor were calculated using Eqs.  and 3)  3) respe31. However, the CV profile remained symmetric without any significant deformation indicating that Co based active material displayed an excellent rate capability.Figure 32.Charge storage mechanism and presence of redox peaks in Fig. 33.Figure 34.CV profiles of Ni based active material deposited on CC in the potential range of − 0.1 to 0.5 V at scan rates of 5 to 100 mV/s are shown in Fig. 35.It was also observed that oxidation peaks shifted to higher potentials while reduction peaks moved to lower potentials with an increase in the scan rates. This can be credited to the limitation of ions diffusion and movement at higher scan rate during the redox reaction. Furthermore, it can be assumed that this phenomenon can be associated with the higher series resistance of NiOIt can be seen from Fig. Similarly, CoNi alloy was also investigated under analogues parameters using CV, GCD and EIS techniques. CV profiles of CoNi alloy at the scan rates of 5 to 100 mV/s in the potential range − 0.1 to 0.5 V are shown in Fig. 2+/Ni3+, Co2+/Co3+ and Co3+/Co4+ associated with OH− to form NiOOH, CoOOH, and CoO2, respectively36. From Figs. 37. Also, at higher scan rates peak potentials shifted and peaks separation increased which is indictive of quasi-reversible nature of electrode materials which is the typical behaviour of pseudocapacitive material38.Pair of these redox peaks around 0.1 and 0.35 V are associated with the reactions Ni39. Furthermore, all GCD profiles also exhibited near symmetric charge–discharge patterns and extremely reversible charge–discharge properties, revealing the superior reversibility of redox reaction along with outstanding capacitive properties. Specific capacitance was calculated from the discharge components of GCD curves and calculated capacitances at different current densities is shown in Fig. 42. However, it can be witnessed that CoNi based active material displayed excellent capacity retention at higher current densities where specific capacitance value dropped moderately from around 176 F/g at current density of 1 A/g to around 122 F/g when current density was increased by around ten times to 10 A/g. It can also be observed that compared with other samples i.e., Co (63 to 45 F/g) and Ni (49 to 25 F/g), drop in capacitance of CoNi sample when current densities are increased from 1 to 10 A/g is substantially lower which can be due to the fact that binary oxides with CoNi being a typical example can afford a richer variety of redox reactions (contribution made by both Ni2+ and Co3+ ions) and possess much superior electrical conductivities compared with monometallic oxide materials43. EIS technique was utilized to analyse the electrical resistivity of CoNi based electrode material and Nyquist plot of this binary compound-based electrode is shown in Fig. 45. CoNi based sample manifested small x-axis intercept in high frequency region  was applied where relationship between current (i) and scan rates (v) was examined46. ‘b’ value is the limiting parameter of the power law equation, b = 1 symbolises surface controlled capacitive process whereas b = 0.5 signifies the diffusion-controlled contribution. Frome the CV profiles of Co, Ni samples from Fig. ‘1’ when compared with Ni sample which implies that the capacitive process is dominated by capacitive behaviour based on surface redox reactions. Furthermore, the charge storage contribution ratios of Co, Ni and CoNi electrodes is measured by evaluating individual charge storage contributions elements conferring to the modified power law,To gain enhanced insight of the overall electrochemical performance and charge storage dynamics of Co, Ni and CoNi electrodes, the power law k1 and k2 are the variables for capacitive and diffusion-controlled processes, respectively47. It can be observed from Fig. Revised toRobustness of CoNi based active material was further examined by cycling through 5,000 charge–discharge cycles at the current density of 10 A/g to evaluate this electrode material for real-world applications. Cell displayed slight degradation with the retention of over 83% of the initial capacitance after 5000 cycles as shown in Fig. Performance comparison for individual samples was conducted in order to understand electrochemical behaviour of each type of active material. Figure S) which is the sum of ionic resistance of electrolyte, intrinsic resistance of electrode, and contact resistance at the electrode/electrolyte interface48. The diameter of the semicircle in the Nyquist plot corresponds to the charge-transfer resistance (RCT) while, in lower frequency region the linear component is associated with Warburg resistance (ZW), which is essentially the resistant component of ionic diffusion/transfer in the electrolyte49. Randle circuit was used as equivalent circuit and Z-view Software was used for EIS plot fitting to obtain RS values where RS values for Co, Ni and CoNi were 0.26 Ω, 0.51 Ω and 0.35 Ω respectively. All sample displayed excellent electrical conductivities with low RS values < 1 Ω where the key contributing factors towards lower RS can be the absence of non-conductive binder and excellent conductivity of carbon cloth. Composite electrode comprising of CoNi with superior capacitive performance has the RS value 0.35 Ω signifying excellent electrical conductivity of hybrid sample. In summary CoNi electrode delivered outstanding charge transfer kinetics demonstrating that the large number of redox reactions can be achieved using this material. Since CoNi has both RC and RT smaller which can result in superior charge transfer kinetics and ultimately large number of redox reactions can be achieved. This can be explained on the basis of band theory as impurity bands can be introduced after mixing these oxide which in turn enhance the electronic conductivity of composites50. These superior electrical properties were also confirmed by cyclic stability and rate capability. Low values of x-intercepts for all the samples means lower equivalent resistance and Warburg component at 45° means lower resistive response and excellent capacitive response48. Magnified Nyquist plot along with equivalent circuit used for fittings is displayed in Fig. EIS was used to analyse resistive/capacitive behaviour of these samples. The Nyquist plots for Co, Ni and CoNi samples are shown in Fig. In summary, we produced Co and Ni oxides, CoNi binary oxides using facile electrochemical deposition technique. By employing this approach Co, Ni and CoNi were directly deposited on highly conductive carbon cloth where carbon cloth was used as both substrate and current collector. During the synthesis of CoNi binary oxides, Co and Ni ratios were fine-tuned by controlling synthesis parameters such as voltage, electrolyte pH, deposition time and electrolyte concentration. The successful deposition of Co, Ni and CoNi was confirmed by number of physical and chemical characterisation techniques. By using this facile technique active material was deposited on carbon cloth and directly used as a binder free electrode in supercapacitor cell. This process provided with an opportunity to produce binder free electrodes with fine-tuned chemical composition. Furthermore, using this process very thin layers of active material can be deposited onto the substrate surface which can be highly useful towards the overall performance enhancement of a SC. Since this can results in the production of light weight and thin electrodes. Reduced weight can result in higher specific capacitance (as given by Eq.


We find that their electronic properties are determined bythe hybridization of the constituent layers and are modulated at themoiré period. The hybridization depends on the layer alignment,and the modulation amplitude vanishes with increasing twist angle.We explain our observations in terms of a hybridization between thenearest sulfur and gold atoms, which becomes spatially more homogeneousand weaker as the moiré periodicity decreases with increasingtwist angle, unveiling the possibility of tunable hybridization ofelectronic states via twist angle engineering.Synthetic materials and heterostructures obtained bythe controlledstacking of exfoliated monolayers are emerging as attractive functionalmaterials owing to their highly tunable properties. We present a detailedscanning tunneling microscopy and spectroscopy study of single layerMoS A famousexample is twisted bilayer graphene, whose very rich phase diagramranges from correlated insulating to superconducting phases dependingon twist angle and electrostatic doping.5 Heterostructureswith unique properties can also be obtained by stacking 2D exfoliatedtransition metal dichalcogenides (TMD).9The rapid increase in the numberof 2D materials which can be exfoliated and the fantastic progressin their layer-by-layer stacking with controlled sequence10 Such structures are challengingto prepare with a clean surface suitable for scanning tunneling microscopy(STM). Therefore, many STM studies to date have been carried out onepitaxial thin films grown in situ by molecular beam epitaxy (MBE)or by chemical vapor deposition (CVD). The twist angle with the substrateof such films is set by thermodynamics and cannot be tuned at will.It is nearly 0° for 2H-MoS2 evaporated on Au(111),13 one of the most studied TMD on a metallic substrate.15Exfoliated 2D materials in directproximity to a metal surfacewith selected twist angles offer further attractive materials engineeringperspectives.2 monolayers (hereaftersimply MoS2) on Au(111) have been published,22 without any strong focus on twist-angle-dependent properties. Here,we present a detailed STM and scanning tunneling spectroscopy (STS)investigation of the electronic properties of MoS2 on Au(111)as a function of the twist angle. High-quality heterostructures allowus to explore genuine twist-angle-dependent properties. We find thatthe system is strongly hybridized independently of the twist angleand that the valence and conductance bands are modulated at the moiréperiod. The modulation amplitude vanishes with increasing twist angle,which we explain in terms of a hybridization between the sulfur andgold atoms that becomes spatially more homogeneous and weaker as themoiré periodicity decreases with increasing twist angle.Manual stacking of exfoliated monolayers allows us to selectarbitrarytwist angles between the 2D material and the metallic substrate. Onlya few STM studies of exfoliated 2H-MoS223 onto template-stripped Au substrates.24 These substrates are polycrystalline Au(111)films stripped from an ultraflat silicon wafer (see SI Section I). ML MoS2 on Au(111)is identified by its characteristic Raman spectrum26 and from optical images where MoS2 appears darker on a bright Au substrate27 (see SI Section I). A further confirmationthat we have ML MoS2 is the observation of a moirépattern in STM topography, which is absent for bilayer and thickerMoS2 flakes.11We performed STM and STS on continuous, millimeter-sized monolayer(ML) flakes obtained by exfoliating 2H-MoSafer see . STM and2 MLs onto polycrystallineAu(111) naturally produces regions with different twist angles betweenthe two lattices. This provides a unique platform to characterizethe electronic structure for different twist angles in a single sample,thus excluding potential sample-dependent fluctuations. 2 lattice and the moiré patterns specificto the local twist angle (SI Section II). The corresponding Bragg and moiré peaks are highlightedin the Fourier transform (FT) of the 7.7° heterostructure bygreen and red circles, respectively . The slight distortionof the moiré pattern in SI Section IV).Exfoliatinglarge area MoSectively 1f. The c2 on Au(111) as a function of the twist angle. Typical I(V) spectra measured on our devices areshown in 28 We do not observe the spreadin tunneling characteristics reported in previous STM experiments,17 most likely due to a cleaner MoS2/Au(111) interface exemplified by the perfect adherence of MoS2 to the substrate across grain boundaries and step edges (SI Section III). We do find occasional bubbles,18 where MoS2 is locally decoupled from the substrate (SI Section V). These regions are electronicallydifferent, with a fully gaped semiconducting I(V) spectrum  tunneling spectra measured on ourMoS2/Au(111) heterostructures, we find some variations,in particularas a function of twist angle and as a function of position in themoiré unit. They are most prominent at negative bias belowthe Fermi level (EF), which correspondsto VBias = 0 V in 2/Au(111) heterostructures,we acquire I and dI/dV maps. For every tunneling conductance spectrum—twotypical examples are shown in EF to a Gaussian and define the valence bandmaximum VBM(r⃗) (conduction band minimum CBm(r⃗)) as the peak position plus (minus) its corresponding2.2σ. Since the conductivity measured at these energies is predominantlyrelated to states derived from MoS2,32 we define Δ(r⃗) = CBm(r⃗) – VBM(r⃗) as thegap. We find that Δ amounts to about 2 eV in agreement withprevious findings for MoS2 MLs grown on Au(111).34 These three quantities are plotted alongside the corresponding topographyfor two different twist angles in I curves using a different fitting procedure (SI Section VI) used in While the generic line shape is the same for all r⃗) is also modulated at the moiré patternwavelength; (iii) the modulation amplitude of VBM(r⃗) and of CBm(r⃗) are decreasing with increasingtwist angle. The vanishing spatial modulations of the band edges andof the gap with increasing twist angle are most strikingly seen in 2 ML, which completely excludes any originother than the twist angle for the observed differences. In r⃗) and CBm(r⃗) (defined as the difference between maximum and minimum band edgeenergies for each twist angle) as a function of twist angle. The plotclearly shows the monotonic reduction of the VBM(r⃗) and CBm(r⃗) modulation amplitudes withan increasing twist angle.The three main results of our experiments can begraphically seenin 2 with Au(111) happens primarily through sulfur p-orbitals and gold d-orbitals, and thatit is strongest when the two atoms are positioned atop each other.35 Calculations furthershow that the hybridization mainly involves out-of-plane orbitals—whichdefine the valence band (VB)—and fewer in-plane orbitals—whichdefine the conduction band (CB).32 Thisis consistent with the greater modulation of the VBM compared to theCBm in 34Density functional theory (DFT) calculationsshow that hybridizationof MoS36 Therefore, it isnot a priori clear whether the moiré superstructure observedby STM is a structural modulation or an electronic modulation. Toaddress this question, we examined dI/dV conductance mapsas a function of bias. The fact that the moiré contrast fullyinverts for certain bias ranges (SI Section VII) and that the moiré modulation amplitude strongly dependson the imaging bias provides strong evidence that the moirépattern is primarily of electronic origin in a very flat MoS2 ML, in agreement with STM and X-ray standing wave measurement bySilva et al.34Constant current STM images are a convolution of morphologicandelectronic features.2 ML on Au(111), we constructa simple model to understand the twist angle dependence of the electronicstructure. We assume that the hybridization is primarily determinedby the nearest neighbor distances (NND) between the S and Au atomsshown as green and yellow spheres in 37 To take thisinto account, we introduce an effective distance deff obtained by convoluting the spatial distribution ofNNDs with a Gaussian. By construction, deff is spatially modulated at the moiré period and provides ameasure of the strength of the local hybridization: it is strongerwhere deff is smaller.Considering a flatMoSdeff, as shown by the histograms in deff. To assess the amplitude of the modulated hybridization,we plot the variance of deff (which reflectsthe width of its distribution) as a function of the twist angle in While the distribution of the distances betweennearest S and Auatoms does not depend on twist angle, their spatial distribution does,with significantly larger site-to-site variations at larger twistangles, as seen in i is a function of the local deff(r⃗i): ΔQi = f(deff(r⃗i)). The average charge transfer isThe average of the VBM and the average of the CBm both shiftdownin energy, with a larger shift for smaller twist angles 4b. This QΔ withinour simple model, we note that althoughthe distribution of deff depends on thetwist angle, its spatial average is constant (SI Section VIII). It means that f(deff(r⃗i)) is not a linear function. We consider two simple nonlinear f(deff(r⃗i)) models which reproduce a strongercharge transfer for the Au–S pairs where deff is shorter. Both models yield the same qualitativeresult.To gain insight into the twist angledependence of 34 in MBE-grown films (0.5°twist angle) thatall the bottom S atoms of the MoS2 ML fall into eitherof two categories: strongly bound or weakly bound to the underlyingAu atom. Thus, we consider two kinds of S atoms: one that contributessignificantly (the strongly bound) and one that does not contributeto the charge transfer (the weakly bound). Assuming a given fraction of significantly contributing S atoms in the 0° twistangle heterostructure, we can estimate a general cutoff deff,c below (above) which all S–Au pairs fall inthe strongly (weakly) bound category with significant (negligible)charge transfer at a given twist angle. In this caseQΔ as a function of twist angle calculated using QΔ monotonically decreases as afunction of the twist angle, which reproduces the observed overalldecreasing charge transfer with an increasing twist angle. We obtainthe same result with another nonlinear function f(deff(r⃗i)) = 1/deff,as shown in SI Section VIII.The first model is motivated by the finding of Silvaet al.2 monolayers onAu(111) using high-resolution scanning tunneling microscopy and spectroscopy.We find that the conduction and valence bands are modulated at themoiré pattern period. The modulations are most prominent inthe valence band and largest for the smaller twist angles. They vanishwith increasing twist angles. We propose a simple model to understandthis twist angle dependence based on a changing hybridization betweenS and Au orbitals, which depends not only on the relative positionsof the nearest S and Au atoms but also on their neighboring configurations.These findings provide detailed insight into designing monolayer-on-metalheterostructures with variable electronic properties and doping byadjusting the twist angle. They also provide a platform to explorecorrelated and ordered electronic phases in combination with periodiccharge transfer (or doping) patterns.In summary,we present a systematic study of the twist-angle-dependentelectronic properties of exfoliated MoS27 onto template-stripped gold substrates38 to mechanically isolate MoS2 monolayers (MLs). The goldsubstrates are prepared in-house. First, we evaporate gold onto aclean ultraflat silicon (Si) wafer. Second, we epoxy another flatSi piece onto the crystalline gold film. We then cleave this sandwichat the evaporated Au–Si interface to get an ultraflat goldsurface that reflects the flatness of the original Si substrate. Bulk2H-MoS2 single crystals were obtained from HQ graphene.These crystals are exfoliated to the monolayer limit onto the freshlyexposed Au surface in a nitrogen-filled glovebox, using scotch-tapeexfoliation. The strong affinity between sulfur and the very cleanand flat template-stripped Au substrate allows us to obtain millimeter-sizedMoS2 MLs. We identify ML MoS2 flakes based ontheir optical contrast on gold and using Raman spectroscopy. A detailedcharacterization of the gold substrate and of the MoS2-on-Auheterostructures is presented in SI Section I and SI Section III. Landing the STM tip on a desired regionon the flake is performed using optical microscopy images. Throughoutthe process, we carefully protect the samples from exposure to theambient atmosphere to avoid contamination and device degradation.For Raman and optical measurements, the samples were placed in a customizedairtight container with optical access that can be sealed in a glovebox.For transferring the samples from the glovebox to the STM, we useda home-built vacuum suitcase which can be directly attached to theload-lock of the ultrahigh vacuum STM chamber. Prior to the STM measurements,the samples were annealed in situ at 150 °C for about 100 h toobtain an optimal surface.We use gold-assistedexfoliation–10 mBar. We used electrochemically etchedW or Ir tips, all carefully conditioned and characterized in situon a Au(111) single crystal. STM topographic images were recordedin a constant current mode. dI/dV(V) conductance curves were acquired by using alock-in with a sample bias modulation amplitude of 15 mV at 527 Hz.All the scanning tunnelingmicroscopy and spectroscopy experiments were done using a Specs JTTyto STM at 77.7 K (0.5° and 21° twisted heterostructures)or 5 K , at a base pressure better than1 × 10R⃗iS/Au) in triangularlattices lying in parallel planes separated by a distance d0 (for a side and top view see dNND(R⃗iAu). Finally, to obtain deff(r⃗i) we spatially convolve the dNND(R⃗iAu) field with a Gaussian kernel. For all further calculations , we discard a small region nearthe edges, i.e. we only consider the region where the convolutionkernel is entirely within the lattice. At every twist angle, we useda large number of atoms (on the order of 106) in each layerto minimize finite size and edge termination effects.For the qualitative modeling of the twist-angle-dependenthybridization, at each twist angle, we first generate the positionsof the S and Au atoms (


Gastrointestinal (GI) microbiome modulators, such as fecal microbiome transplants (FMTs), are being considered as supplements to standard immune checkpoint inhibitor (ICI) treatment to improve efficacy. This systematic review aims to assess the study design and outcomes of clinical trials that use FMTs to enhance ICI treatment.Systematic literature searches were conducted on PubMed and Embase using search terms that included names of ICIs and gastrointestinal microbiome. A first search identified interventional trials, and the second search identified interventional, retrospective, and observational studies.The search for interventional trials produced 205 articles, 3 of which met the inclusion criteria. All studies had sample sizes ranging between 10 and 30 participants. 2 of the studies were single-arm studies with no control arm. One study reported an overall response rate (ORR) of 3 out of 15 (20%), a median progression-free survival (PFS) of 3 months, and a median overall survival (OS) of 7 months. The second study reported 1 complete response out of 10 (10%) and 2 partial responses out of 10 (20%). The third study reported an ORR of 58% vs. 20%, a median PFS of 12.7 months vs. 2.5 months in patients receiving nivolumab–ipilimumab plus CBM588 compared with patients receiving nivolumab–ipilimumab alone respectively, and an undefined median OS.Current studies on the microbiome modulators with ICI use are limited in study design. Future clinical trials should be randomized, use larger sample sizes, and use an appropriate control arm to better ascertain the clinical effect of the GI microbiome on ICI treatment.The online version contains supplementary material available at 10.1007/s00432-023-04656-8. Immune checkpoint inhibitors (ICIs) are commonly used in the treatment of cancers, such as metastatic melanoma, renal cell carcinoma, and non-small cell lung cancer  microbiome  OR “pembrolizumab” [Supplementary Concept]) OR “Nivolumab”[Mesh]) OR “cemiplimab” [Supplementary Concept]) OR “atezolizumab” [Supplementary Concept])) OR “avelumab” [Supplementary Concept]) OR “durvalumab” [Supplementary Concept]) OR “Ipilimumab”[Mesh]) AND “Gastrointestinal Microbiome”“[Mesh]”. The search terms for Embase were ‘immune checkpoint inhibitors’ or ‘checkpoint inhibitor’ or pembrolizumab or keytruda or nivolumab or opdivo or cemiplimab or libtayo or atezolizumab or tecentriq or avelumab or bavencio or durvalumab or imfinzi or ipilimumab or yervoy) and ).mp. .Another systematic search was conducted on April 28, 2022 on PubMed and Embase databases. The search terms for PubMed were “(“Immune Checkpoint Inhibitors”[Majr]) OR "pembrolizumab” [Supplementary Concept]) OR “Nivolumab”[Mesh]) OR “cemiplimab” [Supplementary Concept]) OR “atezolizumab” [Supplementary Concept])) OR “avelumab” [Supplementary Concept]) OR “durvalumab” [Supplementary Concept]) OR “Ipilimumab”[Mesh]) AND “Gastrointestinal Microbiome"[Mesh]”. The search terms for Embase were “ and ).mp. . The searches were limited to clinical trial or randomized controlled trial or controlled clinical trial or multicenter study or phase 1 clinical trial or phase 2 clinical trial or phase 3 clinical trial or phase 4 clinical trial.Since most trials were single-arm trials, we also searched for FDA registration trials for the same tumor type and line of therapy.We included all articles from the first systematic search so that we could categorize study designs.To assess clinical trial design and outcomes in the second systematic search, we included original articles that were in English and were interventional studies using human subjects. We excluded articles that were observational, retrospective, reviews, case reports, and not in English. Additionally, articles that discussed trials on animal subjects, such as mice, were not included due to the variability in study methodology and clinical endpoints between human and animal subjects. Articles were excluded if they were published in abstract form or did not provide sufficient information about the trial. Our primary interest was to focus on the effect of reestablishing the GI microbiome in the efficacy of immune checkpoint inhibitors in the treatment of cancer. Therefore, we excluded trials that focused on interventions that disrupt the GI microbiome, such as antibiotics.For each article identified in the second search, we extracted data that fit into four main categories: 1) article information, 2) patient population, 3) study design and quality, and 4) study outcomes. The article information included author, year published, and NCT number. The patient population included the patient’s diagnosis, cancer type and setting, and demographics of the patient population, such as median age of study participants and percent of male and female participants. Study design and quality factors included the year enrollment began, the intervention and control arms, sample size, endpoints, phase of trial, randomization, blinding, and funding. All results were reviewed by two authors (AB and AH). PRISMA  guidelines were observed.The Jadad score was used to assess the quality of the included controlled trials with a minimum of zero points and a maximum of five points (Berger and Alperson We used US Food and Drug Administration (FDA) data that reported the overall response rate (ORR) for clinical trials that studied pembrolizumab alone and nivolumab alone, respectively, in patients with un-resectable or metastatic melanoma. These data provided us a baseline comparator for outcomes with treatment using the drug alone versus treatment with FMT. A chi-square test was performed to see if there was a significant difference between FDA ORR and FMT ORR. An observed ORR was calculated using both the individual ORR outcomes from the pembrolizumab plus FMT trial and the nivolumab plus FMT trial. The expected ORR was calculated using FDA data on trials using pembrolizumab alone and nivolumab alone. Although the observed and expected outcomes are not directly comparable, the indication for each is similar in studying patients with metastatic melanoma. The chi-square test analysis was conducted using Microsoft Excel, version 16.51. Publicly available data without any patient identifiers were used in our analysis. Therefore, oversight was not required by an Institutional Review Board.n = 42 vs 27, respectively). In PubMed, the next most common article types include prospective observational (n = 22), retrospective observational (n = 5), and mice models (n = 5). In Embase, the next most common study types include disproportionality analyses (n = 21), systematic reviews (n = 13), and retrospective observational (n = 10). Clinical trials were included as one of the least common study types in both PubMed and Embase . Thus, out of the 87 results in PubMed, 2.3% of results were clinical trials and 48.3% were reviews. Out of the 100 results included for Embase, 1.0% were clinical trials and 27.0% were reviews. A visual depiction of this information can be found in Fig. We sought to ascertain the study design of microbiome studies indexed in 2 databases: PubMed and Embase. Up to 100 results were categorized by study design for a representative subset. As PubMed only generated 87 results, all 87 were included in the analysis. The most common study types for both PubMed and Embase were reviews , the average percentage of male population was 71% , and the average percentage of female population was 29% . The remaining study in this analysis did not include demographic characteristics.For studies that reported demographic characteristics of study participants Table , the medClostridium butyricum , and median overall survival (OS). The study utilizing FMT in addition to pembrolizumab for melanoma patients reported an ORR of 20%  is an emerging and revolutionary concept in cancer treatment. In fact, it has already revolutionized the cancer landscape with 43.6% of patients eligible for an IO drug. Below is the link to the electronic supplementary material.