Hereditary hemochromatosis (HH) is associated with an increased risk for hepatocellular carcinoma (HCC) occurring predominantly in patients with cirrhosis at the time of diagnosis. However, HCC in non-cirrhotic liver of patients with secondary hemochromatosis is rare. A 67-year-old man with a history of myelodysplastic syndrome (MDS) was found to have a liver tumor on abdominal computed tomography (CT). On the basis of findings of high levels of serum ferritin and transferrin saturation, and low intensity of liver parenchyma on magnetic resonance imaging (MRI), we made a diagnosis of hemochromatosis. Liver resection was performed and the specimen showed moderately differentiated hepatocellular carcinoma surrounded by non-cirrhotic liver parenchyma with intracellular iron deposition. Multicentric recurrence of liver tumor occurred 2 years after the surgery despite of deferoxamine therapy with well controlled ferritin level. We reported a case of hepatocellular carcinoma in a non-cirrhotic patient with liver iron overload secondary to MDS. It demonstrated the importance of early detection and initiation of treatment of iron overload in preventing HCC in MDS patients, even among Asian population. While Caucasians have a high prevalence rate of hereditary hemochromatosis (HH), the Japanese have a low prevalence rate of the germline HFE mutation because of the rarity of HH in Japan . On the A 67-year-old diabetic Japanese male with a 4-year history of MDS without a history of blood transfusion or ferrotherapy was found to have a liver tumor, which was incidentally detected by computed tomography (CT) when he had pneumonia. He had no contributory family history. The laboratory results indicated macrocytic anemia and normal liver function. Tumor markers including alpha-fetoprotein and PIVKA-II were within the normal limits. The viral markers for hepatitis C and B were negative. The serum ferritin level was high at 2,540 ng/mL, the TIBC and UIBC were 212 and 20 µg/dL, respectively, and the transferrin saturation was 94%.The abdominal ultrasonogram showed a low-echoic mass in the right lobe of the liver, and slightly hyperechoic liver parenchyma. The abdominal CT showed a 5.5-cm solitary low-density tumor in segment 8 of the liver, with contrast enhancement in the arterial phase and washout in the delayed phase. The liver parenchyma showed a density lower than that of the spleen. No radiological evidence of liver cirrhosis was identified. On magnetic resonance imaging, the tumor showed high intensities on the T1-, T2-, and diffusion-weighted image. The liver parenchyma showed prominently low intensities on the T1- and T2-weighted images, indicating extensive iron deposition .per os, which was started after the failure of 5th TACE with progressive disease.On the basis of these findings, the patient was diagnosed with hepatocellular carcinoma and liver iron overload. A resection of segment 8 of the liver was performed without complications. The pathological examination report showed a moderately differentiated HCC without vascular or lymphatic invasion with negative surgical margin. The Mallory stain revealed non-cirrhotic liver with a minimal fibrotic change around the portal vein area. The iron stain showed extensive intracellular iron deposition in the liver parenchyma . A bone Our patient developed secondary liver iron overload due to MDS. Iron overload occasionally occurs in MDS patients owing to ineffective erythropoiesis, followed by increased iron absorption and polytransfusion for anemia. As our patient did not have a history of transfusion or ferrotherapy, increased iron absorption may have played an important role in the occurrence of the iron overload.Although an increased risk of HCC among patients with liver iron overload has been reported, only three cases of HCC associated with secondary iron overload have been reported  5-7] wh-7 wh5-7]Although the prognosis of untreated hemochromatosis is poor, early diagnosis and iron removal therapy improve the survival rate and provide the normal life expectancy of patients . Unfortu


Optical tissue imaging has several advantages over the routine clinical imaging methods, including non-invasiveness (it does not change the structure of tissues), remote operation (it avoids infections) and the ability to quantify the tissue condition by means of specific image parameters. Dermatologists and other skin experts need compact (preferably pocket-size), self-sustaining and easy-to-use imaging devices. The operational principles and designs of ten portable in-vivo skin imaging prototypes developed at the Biophotonics Laboratory of Institute of Atomic Physics and Spectroscopy, University of Latvia during the recent five years are presented in this paper. Four groups of imaging devices are considered. Multi-spectral imagers offer possibilities for distant mapping of specific skin parameters, thus facilitating better diagnostics of skin malformations. Autofluorescence intensity and photobleaching rate imagers show a promising potential for skin tumor identification and margin delineation. Photoplethysmography video-imagers ensure remote detection of cutaneous blood pulsations and can provide real-time information on cardiovascular parameters and anesthesia efficiency. Multimodal skin imagers perform several of the abovementioned functions by taking a number of spectral and video images with the same image sensor. Design details of the developed prototypes and results of clinical tests illustrating their functionality are presented and discussed. Biomedical imaging has become a powerful tool for diagnostics and monitoring of human health condition. Apart from routine clinical imaging modalities , a number of advanced “open air” optical imaging methods and technologies have been introduced recently. Their main advantages are remote operation (avoids infection) and non-invasiveness (does not change the structure of tissues). Besides, digital imaging ensures quantitative documentation on the skin condition and its changes.SIAscope [MelaFind [There are several commercially available skin diagnostic imaging devices for dermatologists , but moMelaFind , video-mMelaFind  and smarMelaFind  have shoMelaFind . This review paper (which follows a previous review ) presentMultispectral imaging is a method based on acquisition of a limited number  of images within relatively narrow non-overlapping spectral bands—so-called spectral images ,12. In tThanks to the rapid development of communication technologies, a number of smartphone camera-based health assessment software applications are available, both in ambient light and under white LED illumination from the same phone ,16. SuchThe system design scheme is presented at DinoLite AD413, series AM-4013) for multispectral imaging by replacing the built-in LEDs with specifically selected color LEDs and by developing the LED management software. A number of digital microscopes nowadays are small handheld devices connected to a PC via a USB cable; they can be applied also for visual skin assessment . A typicDinoLite ring. For better homogeneity of skin illumination, a diffuser film is attached to the new LED-ring. To avoid detection of the directly reflected radiation from the skin surface (thus distinguishing the diffusely reflected radiation from the upper layers of skin), a pair of orthogonally-oriented polarizing filters are added: one of them directly after the diffuser, and the other-in front of the camera sensor matrix. The microscope is computer-controlled. The custom-developed program written in MatLab with a standard FTDI USB driver and a custom LED driver (written in C programming language) performs the control over LEDs and the acquisition of images. The software provides two image processing modes: (1) the preview mode, for focusing the microscope to the skin object, and (2) the video acquisition/processing mode, where the software performs sequential switching of LEDs and triggering of the video sensor. Each single measurement includes capturing of four frames (taken within the four wavelength bands). After recording, the images are stored in a 4-image matrix and saved as a data file for further processing. The image processing software allowed mapping of three above-mentioned skin chromophores, erythema index and the melanoma/nevus differentiation parameter [The custom-designed control unit module is installed on the rear side of microscope as shown on arameter . More dearameter ,23,24.If skin is evenly illuminated by several laser sources, monochromatic spectral images can be extracted from a single RGB image file , thus ma1, λ2 and λ3  and the adjacent healthy skin (ojI). The ratios Ij/Ijo at each pixel or pixel’s group of three monochromatic spectral images contain information on the concentration increase or decrease of all three regarded chromophores, which can be further mapped over the whole image area [The general concept of snapshot skin chromophore mapping at fixed wavelengths is illustrated at age area . PGL-DF-450nm-20mW-15011564, PGL-VI-1-532nm-20mW-15030443 and PGL-DF-655nm-20mW-150302232 from Changchun New Industries Optoelectronics Tech. Co., Ltd., Changchun, China). Laser modules , i.e., self-fluorescence without any specific additives on or inside the skin. There is a number of fluorescing compounds called fluorophores in the upper skin, each with its specific emission spectrum. Even if excited by a narrow laser line, several emission spectra overlap and skin autofluorescence spectrum usually is bell-shaped, without a pronounced structure. Besides, a phenomenon called autofluorescence photobleaching (AFPB) normally takes place: the in-vivo skin emitted intensity decreases during continuous optical excitation and does not fully recover after interruptions of the excitation . AFPB caI—autofluorescence intensity at a fixed wavelength band, t—time, a and b—weighting coefficients, A—the “bottomline” constant, and τ1, τ2—the “fast” and “slow” AFPB rate coefficients, respectively. The “fast” AFPB (when sharp intensity decrease is observed) usually takes place during the first 5–15 s of the irradiation, while the “slow” AFPB continues up to several min.The decrease of skin autofluorescence intensity during the laser exposure in most cases can be approximated by a double-exponential expression:Our first set-up for skin AFPB imaging comprised laser illuminator and consumer photo camera equipped with a band-pass filter in front of the objective; it was operating at a slow video-mode (~2 frames/s) . After t2 with framerate 0.5 fr/s is recorded for 20 s. Four battery-powered violet LEDs placed within a cylindrical light-shielding wall  evenly irradiate a 40 mm round spot of the examined skin tissue. A long pass filter (>515 nm) is placed in front of the smartphone camera to prevent detection of the exciting LED emission. The battery/electronics compartment comprises a set of rechargeable batteries and a Bluetooth Low Energy (BLE) module with a driver for communications between the smartphone and illumination unit. As the next step, a smartphone-compatible technique for acquisition and analysis of violet LED excited skin fluorescence intensity and AFPB rate distribution images has been developed and clinically tested . Design Samsung Galaxy Note 3 smartphone comprising integrated CMOS RGB image sensor with resolution of 13 MP was used for image acquisition. All images were taken at the following settings: ISO-100, white balance—daylight, focus—manual, exposure time—fixed 200 ms. More details on the skin fluorescence imager design and its test results are provided in [The recorded RGB fluorescence images were further separated to exploit the R- and G-images for imaging of skin autofluorescence in the red and green spectral bands, respectively. Due to spectral cut-off by the 515 nm long pass filter, the B band images served only for reference. A vided in ,39.The incident cw light can be reflected from the skin surface and also may enter its epidermal and dermal layers where light can be absorbed and/or scattered. A part of back-scattered photons have been travelled via the skin dermal layer where arterial blood volume periodically changes with each heartbeat. As a consequence, also total blood absorption changes with each heartbeat and the back-scattered light intensity is modulated—the detected so-called remission photo- plethysmography (PPG) signal comprises a relatively stable DC component, determined by absorption of the “static” skin structures, and a pulsatile AC component caused by the periodically changing blood absorption . The puluEye software using manual trigger mode, fixed exposure time, 2 × 2 pixels binning and triggered at 60 frames/s. The monochromatic camera (8 bit CMOS IDS-uEye UI-1221LE) was equipped with an S-mount 1/2 inch F = 4 mm low distortion, wide-field lens . The camera lens was placed at 15 cm distance from the skin surface so ensuring full view of adult palm (20 cm × 15 cm field of view). In order to reduce skin specular reflectance, orthogonally oriented polarizers were placed behind the camera and all four light sources, respectively. The plastic parts were produced by a 3D printer . The device comprised a plastic enclosure filled with an adjustable vacuum pillow  as the palm support.The microcontroller board  provided sequential switching of green and IR LEDs and triggering of the captured video frames. The camera control was performed by IDS uEye UI-1221LE), resolution 752 × 480 pixels, maximum framerate 87 fps., 8-bit monochrome). The camera is equipped with low-distortion S-mount 4 mm lens (Lensagon). In order to reduce skin specular reflectance, cross-oriented polarizers are placed behind the light sources and in front of the camera, respectively. The infrared cut-off filter KC-15 (>700 nm) also is placed in the front of the camera, in order to minimize the influence of ambient illumination. USB-2 cable connection to PC ensures both video-signal processing and the power supply. Another, much smaller PPGI prototype device  for moreFor real clinical applications, it is a good choice to use surgical lamp as a light source. A simple and convenient PPGI system for contactless monitoring of anesthesia effectiveness before and during surgical procedures has been developed . The sysA proof-of-concept prototype device based on inexpensive components and smart software was developed for compact and handy wireless skin diagnostics and monitoring. The multimodal imaging includes capturing by a single camera a number of spectral and video-images from the skin pathology area, with subsequent extraction of clinically significant information. The device captures four consecutive imaging series: (i) RGB image of skin at white polarized LED illumination, helping to reveal hidden subcutaneous structures; (ii) four spectral images at narrowband LED illumination for mapping of the main skin chromophores; (iii) video-images under green LED illumination for mapping of skin blood perfusion; (iv) autofluorescence video-images under UV LED irradiation for mapping of the skin fluorophores. Polarized LED light is used for illumination, and round skin spot of diameter 34 mm is imaged by a CMOS sensor via cross-oriented polarizing filter. To improve the reliability of diagnostics, manipulation with maps of different parameters  is proposed, as well. Our first prototype version was described earlier . SkImager [Tegra 2 T20  system on chip (SoC) module with a dual-core Cortex-A9 processor  is used as a central processing unit. It provides smooth operation of all components of the device. A 3 Mpix RGB CMOS matrix with 3.2 micron pixel size (MT9T031) serves as the image sensor; it is connected to the central processor via 10-bit parallel line. Removable SD memory card stores the image information that can be transferred to external processor, e.g., PC. It can be done also via Mini-USB connector which also ensures software installations and updates from outside. On-board and off-board calculations can be performed to extract parametric maps of the examined skin area. The main information input-output device is the built-in 4.3 inch/480 × 272 pixels touchscreen. It displays the operation mode and state of the device . Power switch button is placed on the side above the slot of memory card  module uses embedded computer—Raspberry Pi  as the mThe third (illumination) module ensures three imaging modes: multispectral imaging of diffuse reflectance , fluorescence spectral imaging (excitation at 405 nm) and 635 nm laser speckle imaging. Custom printed board was created for managing skin illumination by narrowband LEDs. It uses standard interface for selecting current illumination band, therefore light source change requires less effort than in fully customized existing designs.By using her/his own smartphone or laptop, the user connects to the device’s WiFi network and controls imaging process through the Internet browser. During the first step, the operating system’s procedure for connecting to the WiFi network (with a password-controlled access) is performed. On the step two users selects whether he/she requires a new image capture or wishes to view the previous results. During the third step user targets the skin region to be captured by viewing live video stream from the camera. Image is captured by pressing a physical button located on the device. The capturing process can be repeated a number of times. After all skin images are acquired, user can switch to the last—results viewing step. Typical time delay between finishing the image capture procedure and obtaining the results is less than 10 s. More details on this device and its tests are presented in .The developed skin imaging prototypes were initially tested in laboratory and then in real clinical environments. All clinical studies were performed with written consent of the involved volunteers under official approval by the local ethics committee. Some results of the clinical measurements, aimed at checking functionality and appropriateness of the prototypes, are briefly presented below.NEXUS5 smartphone camera, in comparison with spectral images of another nevus, taken under similar illumination by the SkImager prototype , green (G) and blue (B), respectively. The values of RGB components were defined from the image data by a special program developed in MATLAB®.Overall 50 patients with 150 different skin neoplasms were inspected with the smartphone based fluorescence imager. For more detailed image analysis 13 basal cell carcinomas (BCC) and 1 atypical nevus were selected . In ordeIn all BCC cases  the fluorescent images showed lowered AF intensity in malignant tissue as compared with the healthy surrounding skin, which may be attributed to lower concentration of fluorophores and increased blood perfusion caused by the malignant process. A case of solid BCC is illustrated in The examined atypical nevus  before sIn order to evaluate ability of the double-wavelength PPGI prototype  to discrThe subjects were seated on a comfortable reclined chair with the right hand at the dorsal aspect, fixed on the vacuum pillow support b and kepLocal anesthesia affects the sympathetic vascular tone, resulting in vasodilation and raised skin blood perfusion. This increases the amplitude of fast-varying signal detected by the PPGI prototypes—see The principles of operation and design features of ten recently developed prototype devices for diagnostic skin imaging have been presented, along with some clinical test results confirming their practical applicability. The laboratory-made prototypes were mainly intended as proof-of-principle devices, able to examine suitability of the developed design concepts and software solutions for further implementation in real clinical environments. Diagnostic sensitivity/specificity, influence of skin color, epidermal thickness and/or optical clearing have not been considered at this stage, as they can be evaluated only after statistically representative clinical trials.Tests of the multispectral skin imaging prototypes led to conclusions on critical conditions to be met for successful extraction of skin parametric maps  from the captured spectral images. The most important ones are uniformity of skin illumination at all used spectral bands, long-term repeatability of all spectral illumination parameters, possibly narrow spectral bandwidths  and short image acquisition time (single snapshot mode preferred). The developed designs are intended for imaging of relatively flat skin areas; the curved ones  still remain an issue due to uneven illumination and varying focal distance. Speckle free multi-laser illumination  seems toFluorescent skin imaging is relatively less complicated non-contact diagnostics technique. It was shown that even a simple and inexpensive smartphone-illuminator system  can provThe developed photoplethysmography imaging prototypes and software have revealed new challenges for distant non-contact monitoring of skin blood perfusion changes at different vascular depths and under anesthesia. The latter application seems to be most successful if the camera is attached to the surgical lamp or originally integrated there. Stabilized DC power supply to the lamp is preferred, but the AC supply also does not influence the results much if the video frame rate is selected equal to the light pulsation frequency. Fusion of PPGA images with real body images  seems toTests of the developed multimodal imaging prototypes have confirmed their proposed functionality. However, from the point of clinical users the number of offered options seems to be too high and more specialized devices based on the tested concepts eventually might be more successful in the medical device market.Implementation of new design ideas and practical tests of the prototypes will promote better understanding of the challenges and drawbacks of in-vivo skin imaging technologies. Their clear advantages are remote and nearly real-time operation, as well as ability of quantitative pathology documentation. On the other hand, image-based clinical criteria for skin pathology diagnostics either do not exist or are in very early stage of development. Routine healthcare needs “golden standards” for fair assessment of patient’s condition and for setting up the recovery strategy, and from this point optical imaging techniques still have to be checked at numerous clinical measurements before becoming a standard of care. Even if some of the above-described skin imaging prototypes would appear clinically acceptable, lots of efforts will be needed in future to establish and validate specific image parameters related to clinically significant threshold levels of particular skin pathologies.


The human immune system is a fine network consisted of the innumerable numbers of functional cells that balance the immunity and tolerance against various endogenous and environmental challenges. Although advances in modern immunology have revealed a role of many unique immune cell subsets, technologies that enable us to capture the whole landscape of immune responses against specific antigens have been not available to date. Acquired immunity against various microorganisms including host microbiome is principally founded on T cell and B cell populations, each of which expresses antigen-specific receptors that define a unique clonotype. Over the past several years, high-throughput next-generation sequencing has been developed as a powerful tool to profile T- and B-cell receptor repertoires in a given individual at the single-cell level. Sophisticated immuno-bioinformatic analyses by use of this innovative methodology have been already implemented in clinical development of antibody engineering, vaccine design, and cellular immunotherapy. In this article, we aim to discuss the possible application of high-throughput immune receptor sequencing in the field of nutritional and intestinal immunology. Although there are still unsolved caveats, this emerging technology combined with single-cell transcriptomics/proteomics provides a critical tool to unveil the previously unrecognized principle of host–microbiome immune homeostasis. Accumulation of such knowledge will lead to the development of effective ways for personalized immune modulation through deeper understanding of the mechanisms by which the intestinal environment affects our immune ecosystem. The jawed vertebrates have evolutionally acquired a unique immune system consisted of effector and regulator cells that can effectively respond or establish tolerance to millions of endoneous and environmental antigens in an epitope-specific manner . In thisDuring the past few decades, accumulative lines of evidence have indicated that the host microbiome and nutrients not only play key roles for balancing the host immunity in health and disease but have a tremendous influence on the generation and shaping of immune cell repertoire , 3. For Clostridia species but flexible diversity of the host TCR repertoire. Transgenic mice genetically engineered to express a restricted TCRβ repertoire spontaneously developed severe colitis in association with hyperactivation of T helper 17 cells (Th17) and a striking decrease in a special subset of peripherally derived Tregs responsible for the recognition of intestinal microbiota  and its ligand , 8. Analclerosis , 14, sugIt is also well known that “the first microbial gut colonizers” play an essential role for the development and shaping of the early immune system in neonates and infants . For insCollectively, these observations strongly indicate that the interaction of gastrointestinal tract and indigenous microbiota is a life-long key regulator of well-balanced immunity and tolerance possibly by shaping adaptive immune cell repertoire.TRB (TCRβ) and IgH loci in a given lymphocyte population. Since these pivotal studies, a rapidly increasing number of researchers have installed NGS-based high-throughput sequencing of TCR (TCR-seq) and BCR/immunoglobulin (BCR/Ig-seq) clonotypes to elucidate the characteristics and dynamics of immune repertoire in healthy individuals and patients with immune dysregulation  or V-J  gene segments, (ii) massively parallel sequencing of the PCR amplicons, and (iii) allignment of NGS reads by use of sophisticated bioinformatic technologies. However, many technical caveats still exist in these approaches  PCR amplification of V-D-J , consisted of only a few thousands TCRα and TCRβ clonotypes per individual, also contains such “shared” fractions . Intriguingly, zebrafish T cells bearing shared TCRs are reported to show predominant expansion in response to diverse antigenic stimuli, suggesting high crossreactivity of these public T cell clones in system-wide T-cell repertoires of teleost fish . Furthermore, these microbiota-reactive CD4+ T cells were polarized to IL17A+ single producers after treatment with inflammatory cytokine cocktails or in patients with inflammatory bowel diseases, suggesting that some of these CD4+ T cell clones function as effectors rather than bystanders at least in selected gastrointestinal inflammatory conditions  and was further enriched in the intestinal tissues . Also ofnditions .Similarly, the majority of IgA- or IgG-expressing memory B cell repertoires residing in human terminal ileum are reported to possess antigen-specificities for the representatives of commensal flora and intestinal pathogens , suggestin silico study using proteome datasets has reported the extensive sharing of possible T-cell exposed peptide motifs between human proteome and gastrointestinal microbiome (Bacteroides were shown to be a mimotope of an established pancreatic β cell autoantigen in non-obese diabetogenic mice (Bacteroides in germ-free mice demonstrated that the recruitment of diabetogenic CD8+ T cells in a microbial epitope-dependent manner. Surprisingly, these crossreactive T cells consistently express an invariant TCRα chain and function to protect the host mice from experimental colitis, suggesting that TCR repertoire of effector and regulatory T cells might be inherently crossreactive. Accordingly, the classical dichotomy of immunologic “self” and “non-self” should be revised and redefined because it is difficult to clearly distinguish “genetic self” and “non-genetic/microbial self” (T-cell crossreactivity is an important area of ongoing researches in system immunology and probably one of the not yet fully recognized chief principles of adaptive immune response. Consistent with this hypothesis and in contrast to the previous general belief, an elegant study using TCRβ transgenic mice proved that the vast majority of negatively selected TCRs are autoreactive and endowed with crossreactivities against multiple MHC haplotypes, while crossreactive TCRs are very infrequent among preselection TCRs . In thiscrobiome . Very renic mice . Monocolal self” . NGS-basClostridium and Akkermansia. In addition to well-established mice models, recently adapted gnotobiotic zebrafish husbandry may offer advantages because it can facilitate system-wide analyses at lower experimental costs, although there are still challenges in translation of the experimental results into humans (During the past decade, rapid innovations in genomic and bioinformatic technologies in the interdisciplinary field of microbiology and immunology have radically changed the outlook of human host–microbiome interactions and their influences on human health and disease. In particular, advanced methodologies in high-resolution adaptive immune repertoire analysis will provide an essential clue to obtain deeper understanding of the ontogeny of our immune system with its microbiome fingerprints at the individual level. Given these backgrounds in mind, it is very attractive to identify and trace the members of adaptive immune cell repertoire by single-cell TCR-seq and BCR/Ig-seq in mono- or poly-colonized germ-free animal models. Such studies will reveal the potential full diversity of TCR and BCR/Ig repertoire created by antigenic epitopes derived from intestinal commensals of particular interest, such as o humans . AnotherThe studies by the authors included in this manuscript were approved by the animal experiment committee and the ethical committee of Hiroshima University.TI and TM contributed equally to this work. All authors listed have made substantial contributions to text and have approved the final manuscript for submission.RS is the chief executive director of Repertoire Genesis Incorporation. KK and HS are employees of Repertoire Genesis Incorporation. TS is the chief executive director of BITS Co. Ltd. No other authors have relevant conflicts of interest to declare.


Non-viral and viral vector mediated delivery have been developed for temporary or persistent expression of CRISPR components in retinal cells. Although in theory CRISPR-based genome editing can correct a large number of mutant genes responsible for a variety of inherited retinal disorders (IRDs), precise gene modification relies on homology-directed repair (HDR)–the efficiency of which is not currently high enough for meaningful benefit. Development of CRISPR-based treatment for retinal diseases thus far has been mainly focused on gene knock-out or gene deletion in which the highly efficient non-homologous end joining (NHEJ) repair pathway is involved. Therapeutic benefits have been achieved in a few rodent models of retinal diseases following CRISPR treatment. The in vivo applications of CRISPR have also facilitated studies of gene function in the retina. As off-target events and immune responses are still the major concerns, continuous development of safer CRISPR genome editing systems is prerequisite for its clinical applications.The rapidly evolving CRISPR-based genome editing technology is bringing revolutionary changes to the entirety of the life sciences. In this mini-review, we summarize the recent progress of  Genome editing is a group of techniques used to modify the genome of a cell or organism. It usually involves the introduction of an engineered nuclease leading to the generation of a double strand break (DSB) at a desired location on the genome, followed by an endogenous DNA repair process through non-homologous end joining (NHEJ) or homologous recombination (HR) Figure . NHEJ ishttps://sph.uth.edu/retnet/sum-dis.htm), which could be treated by gene intervention including genome editing. Gene intervention could also modulate the disease pathways of multifactorial retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy, and glaucoma. Compared to the gene replacement (or gene augmentation) approach that is commonly adopted for treatment of recessive diseases, precise gene repair by genome editing is more attractive as it may permanently restore endogenous gene expression. Meanwhile, gene disruption by genome editing could be more efficient than the RNA interference–based approach for treatment of dominant diseases. However, in vivo genome editing in the retina was rarely conducted before the emergence of the CRISPR-based technology. This was not only because of the complexity in designing sequence-specific nucleases including meganucleases, ZFNs and TALENs, but also because of their large coding sequences hard to be delivered into retinal cells. In contrast, the Cas9 endonuclease gene, either alone, or together with the small guide RNA (sgRNA) expression cassette, is small enough to be packaged into one or two adeno-associated viral (AAV) vectors that are capable of transducing retinal cells efficiently. In this regard, in vivo application of CRISPR/Cas9 genome editing in the retina, though only at its infant stage, represents a significant step forward in developing new treatments for retinal diseases. In addition, for gene function studies, it provides an alternative to gene-knockout by the postnatal intervention of the genome. Although several reviews on similar topics have been published  are caused by mutation (s) in one or more of over 200 different genes or loci  following subretinal injection of a plasmid encoding the CRISPR components was used in a few studies and efficient genome editing was detected in transfected retinal areas  approaches the packaging limit of an AAV vector, a dual vector system with one vector carrying the SpCas9 and the other carrying the single-guide RNA (sgRNA) is usually employed. A successful knockout of YFP gene in mouse retinal cells was reported using AAV type 2 (AAV2)-mediated CRISPR delivery   , a severe retinal degenerative disease with early onset and rapid progression. The most frequent mutation found in patients with CEP290-LCA is a deep intronic mutation (c.2991 + 1655A > G) in intron 26 of the CEP290 gene (IVS26 mutation) that generates a cryptic splice donor site. A recent study showed that a pair of sgRNAs coupled with SpCas9 were highly efficient at removing the IVS26 mutation and restoring the expression of wild-type CEP290 in 293FT cells introduced with the mutation  gene encoding a transcription factor that specifies the rod cell fate. Following AAV8-mediated CRISPR delivery to the mouse retina, NRL expression was markedly reduced in rods, resulting in a gain of certain cone features and partial loss of rod function. The transduced rods presented with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. Our results suggest that the CRISPR-mediated NRL disruption in rods could be developed into a viable treatment option for patients with retinal degenerative diseases   agents has been an important treatment strategy for AMD and diabetic retinopathy, but current approaches require repetitive administration. To develop a long-term solution, two studies from a same group reported the disruption of the mouse Mertk gene is deleted leading to retinal degeneration, the HITI approach allowed the successful insertion of exon 2 into intron 1, resulting in partial restoration of MERTK expression, retinal morphology and function  may broaden the scope of retinal diseases that could be treated by CRISPR. HITI relies on the NHEJ pathway to achieve gene knock-in at the desired genome location in both dividing and non-dividing cells Figure , with anin vivo gene disruption or deletion is more rapid and cost-effective, providing an alternative for gene function studies. To investigate the function of a 108 bp fragment  in the 5′ untranslated region of Blimp1, a transcription factor that regulates the rod vs. bipolar cell fate decision, Wang et al. applied CRISPR to delete the B108 fragment in retinal cells by electroporation to neonatal mice  recognizable by a specific recombinase  needs to be genetically engineered to flank the target gene. Compared to conditional gene knock-out, CRISPR-mediated post-natal in vivo expression CRISPR components in humans following vector delivery, but also induce tissue or organ damage due to cytotoxic T cell response. However, CRISPR applications in the retina might be able to avoid the pre-existing immune responses if subretinal vector administration is used, as the subretinal space is segregated from the blood circulation. Nevertheless, cautions should be exercised, and more work needs to be done before CRISPR genome editing can be used in vivo in humans.As the CRISPR components originated from prokaryotic cells, their expression may cause cytotoxicity and trigger immune responses in mammalians. Although persistent expression of CRISPR components did not cause obvious tissue damage in previous studies including our own   sequences have greatly expanded the number of target sites available for genome editing (Komor et al., e Figure .All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The grasp reflex is a distressing symptom but the need to treat or suppress it has rarely been discussed in the literature. We report the case of a 17-year-old man who had suffered cerebral infarction of the right putamen and temporal lobe 10 years previously. Forced grasping of the hemiparetic left upper limb was improved after a unique combined treatment. Botulinum toxin type A (BTX-A) was first injected into the left biceps, wrist flexor muscles, and finger flexor muscles. Forced grasping was reduced along with spasticity of the upper limb. In addition, repetitive facilitative exercise and object-related training were performed under low-amplitude continuous neuromuscular electrical stimulation. Since this 2-week treatment improved upper limb function, we compared brain activities, as measured by near-infrared spectroscopy during finger pinching, before and after the combined treatment. Brain activities in the ipsilesional sensorimotor cortex (SMC) and medial frontal cortex (MFC) during pinching under electrical stimulation after treatment were greater than those before. The results suggest that training under electrical stimulation after BTX-A treatment may modulate the activities of the ipsilesional SMC and MFC and lead to functional improvement of the affected upper limb with forced grasping.  The grasp reflex is a primitive reflex that may reappear in the presence of lesions in the frontal lobe. Involuntary movements and dystonia are well-known features of neurodegenerative diseases in the basal nucleus. They are both distressing symptoms and have specific impacts on the quality of life of patients. There are several effective drugs for involuntary movements, such as L-DOPA, trihexyphenidyl hydrochloride, clonazepam, and botulinum toxin type A (BTX-A) , 2. HoweBTX-A is a very effective treatment for spasticity and dystonia in patients with stroke, cerebral palsy (CP), or spinal cord injury (SCI) . To our The objective of this case report is to describe the use of BTX-A treatment accompanied by both repetitive facilitative exercises (RFE) and object-related training under low-amplitude continuous neuromuscular electrical stimulation (NMES) for a patient with the grasp reflex after infarction of the right putamen and temporal lobe. The patient showed functional improvement after BTX-A treatment. The change in brain activity was examined using near-infrared spectroscopy (NIRS).This was a single-patient case study. A 7-year-old boy with right cerebral infarction suffered from left hemiplegia, forced grasping, and unilateral spatial neglect. Brain CT scan revealed cerebral infarction of the right putamen and temporal lobe . Two monHe was admitted again for BTX-A treatment at 4 months after his discharge. The severity of left hemiparesis BRS was 5 in the upper limb, 3 in the hand, and 5 in the lower limb. Forced grasping and involuntary movement of the upper limb were so remarkable that he could not use tools with his left hand. The Simple Test for Evaluating Hand Function (STEF) score on admission was only 8 points. The STEF was designed to evaluate the speed of manipulation (catching or pinching objects and carrying them) of objects (10 different shapes and sizes) using an upper limb . The maxThe patient was treated by the injection of 200 units (U) of BTX-A  under electromyography (EMG) guidance soon after admission. BTX-A was injected into the following muscles: left biceps (50 U), flexor carpi radialis (25 U), flexor carpi ulnaris (25 U), flexor digitorum superficialis (35 U), flexor digitorum profundus (25 U), flexor pollicis longus (20 U), flexor pollicis brevis (10 U), and adductor pollicis (10 U), respectively. One week after the BTX-A injections, the spasticity of the left upper limb and the fingers improved and the grasp reflex and involuntary movement nearly disappeared . He coulRFE were applied after the BTX-A injections. RFE were designed to elicit and maintain movements isolated from synergy, including the movement of each isolated finger using a stretch reflex, skin-muscle reflex, and alpha-gamma linkage. The hypothesized mechanism of the newly designed facilitation exercises for the fingers is shown in μs and frequency of 20 Hz. The intensity of the electrical current was adjusted to produce slight contraction of the target muscle without inducing obvious limb/joint movement while the patient remained at rest and was subjectively comfortable. He repeated the training to grasp and release the object under NMES. RFE were applied under NMES ) and the concentration of deoxygenated hemoglobin ([deoxy-Hb]) using 3 wavelengths  according to the Beer-Lambert law. These [oxy-Hb] values reflect not only the hemoglobin density but also thepath length of light. Each channel is configured by a pair of source/detector probes, which are separated by a distance of 30 mm. The channel is supposed to be set at the midpoint between the two probes 20~30 mm under the scalp see .The source probe emitted light sequentially to avoid cross-talk noise, and the sampling time was adjusted to 0.1 s which was sufficiently fast to measure the fluctuation.The time course data were acquired at each channel during the pinching test. The patient sat on a chair during the experiment. The patient was instructed to relax for 10 s, and then the operator ordered the patient to start pinching with the left thumb and index finger every 2 s at the operator's command. After 20 s of pinching, the patient was instructed to relax for 10 s; 5 cycles of this 10 s rest, 20 s pinching task, and 10 s rest were performed for averaging . Two conT values greater than 2.3 ) in the ipsilesional SMC after BTX-A treatment combined with NMES compared with that before BTX-A treatment Figures .When continuous NMES was applied to the extensor side of the forearm during pinching, there was activation in both SMC and ipsilesional prefrontal cortex (PFC) before BTX-A treatment combined with NMES . The actIn a comparison of the activation area after BTX-A treatment combined with NMES, the activation area with continuous electrical stimulation of the extensor side of the forearm  was smalThe grasp reflex and involuntary movement after cerebral infarction improved with a decrease in spasticity by BTX-A treatment combined with both RFE and object-related training under continuous NMES. NIRS detected an increase in blood flow in the right cerebral motor cortex during left hand pinching after BTX-A treatment combined with NMES. More regional activity of the right motor cortex was detected during continuous electric muscle stimulation of the extensor side of the forearm.The grasp reflex can be elicited in neonates and early infants as a result of insufficient control of the spinal mechanism by the immature brain, but the reflex gradually disappears as the infant grows, due to increased inhibition accompanying brain maturation . Adult pIn a study by De Renzi and Barbieri , the palIn our patient, it seems that inhibitory stimuli from upper brain structures were obstructed by lesion of the right putamen to release the spinal grasp reflex center.A major role of the basal ganglia could be to achieve a balance between excitatory and inhibitory thalamocortical influences. The basal ganglia appear to “gate” sensory input at various levels . There iIntramuscular injection of BTX-A was performed to treat muscle spasticity and dystonia. The therapeutic effects of BTX-A seemed to be due not only to partial denervation of extrafusal muscles but also to fusimotor denervation of intrafusal fibers that tonically control the sensitivity of spindle sensory afferents –21. ConsContinuous electrical stimulation of the wrist and finger extensor muscles after BTX-A treatment could also inhibit antagonistic muscles such as the flexor muscles of the wrist and finger. Brain activity in the ipsilesional SMC and MFC areas was more improved with continuous electrical stimulation while pinching, as examined by NIRS. Furthermore, activities in the contralesional hemisphere during continuous electrical stimulation appeared to be suppressed after BTX-A treatment . This suIn our patient, we considered that the frontal lobe did not suppress the grasp reflex or involuntary movement induced by sensory input to the left hand. However, BTX-A treatment combined with NMES improved voluntary movements. This improvement may have been due to not only a decrease in peripheral spasticity, but also a decrease in the excitement from the muscle spindle to the afferent nerve fibers to the spinal cord, and this indirectly influenced the cerebral cortex and improved the cerebral balance to make it easy to perform voluntary movement. Electrically mediated repetitive movement may facilitate the neuroplasticity of motor learning.The grasp reflex of the hemiplegic left upper limb of a patient who suffered infarction of the right putamen and temporal lobe was significantly improved by BTX-A treatment combined with both RFE and object-related training under NMES. This improvement of the grasp reflex by BTX-A consisted of not only the improvement of spasticity but also the improvement of motor control of finger movement. Further research on BTX-A treatment combined with NMES is needed in patients with the grasp reflex that is not improved with ordinary treatment.


In colorectal cancer (CRC), unresectable liver metastases are associated with a poor prognosis. The FOXFIRE , SIRFLOX , and FOXFIRE-Global  clinical trials were designed to evaluate the efficacy and safety of combining first-line chemotherapy with selective internal radiation therapy (SIRT) using yttrium-90 resin microspheres, also called transarterial radioembolization.The aim of this analysis is to prospectively combine clinical data from 3 trials to allow adequate power to evaluate the impact of chemotherapy with SIRT on overall survival.Eligible patients are adults with histologically confirmed CRC and unequivocal evidence of liver metastases which are not treatable by surgical resection or local ablation with curative intent at the time of study entry. Patients may also have limited extrahepatic metastases. Final analysis will take place when all participants have been followed up for a minimum of 2 years.Efficacy and safety estimates derived using individual participant data (IPD) from SIRFLOX, FOXFIRE, and FOXFIRE-Global will be pooled using 2-stage prospective meta-analysis. Secondary outcome measures include progression-free survival (PFS), liver-specific PFS, health-related quality of life, response rate, resection rate, and adverse event profile. The large study population will facilitate comparisons of low frequency adverse events and allow for more robust safety analyses. The potential treatment benefit in those patients who present with disease confined to the liver will be investigated using 1-stage IPD meta-analysis. Efficacy will be analyzed on an intention-to-treat basis.This analysis will assess the impact of SIRT combined with chemotherapy on overall survival in the first-line treatment of metastatic CRC. If positive, the results will change the standard of care for this disease.FOXFIRE ISRCTN Registry ISRCTN83867919; http://www.isrctn.com/ISRCTN83867919 (Archived by WebCite at http://www.webcitation.org/6oN7axrvA). SIRFLOX ClinicalTrials.gov NCT00724503; https://clinicaltrials.gov/ ct2/show/NCT00724503 (Archived by WebCite at http://www.webcitation.org/6oN7lEGbD). FOXFIRE-Global ClinicalTrials.gov NCT01721954; https://clinicaltrials.gov/ct2/show/NCT01721954 (Archived by WebCite at http://www.webcitation.org/ 6oN7vvQvG). The 5-year overall survival (OS) of metastatic colorectal cancer (mCRC) patients, who constitute 21% of all CRC diagnoses, is approximately 13% -8. The uAmong the liver-directed therapies that may control liver metastases, selective internal radiation therapy (SIRT) is one option for patients with liver-only or liver-dominant disease ,12. SIR-The FOXFIRE , SIRFLOX , and FOXFIRE-Global  clinical trials were designed to study SIRT in combination with chemotherapy, specifically the modified fluorouracil, leucovorin, and oxaliplatin (FOLFOX) regimen, compared with FOLFOX alone as first-line therapy for mCRC ,14. EligThe primary objective of the combined analysis is to report the planned meta-analysis (MA) of individual participant data (IPD) from the FOXFIRE, SIRFLOX, and FOXFIRE-Global studies for the primary endpoint, OS, and for secondary outcomes including PFS, liver-specific PFS, quality of life measures, response rate, resection rate, and adverse event profile. The IPD-MA will assess the treatment effects on clinical outcomes in the entire subject group and in key subgroups.As the results on survival benefits for each of the 3 studies are still blinded, the statistical design is a prospective MA based on randomized IPD from the FOXFIRE, SIRFLOX, and FOXFIRE-Global clinical trials.The protocols of the FOXFIRE  and SIRFAll randomized patients from the SIRFLOX, FOXFIRE, and FOXFIRE-Global clinical trials will be included in the combined analysis.Histologically confirmed CRC with liver-only or liver-dominant metastases not amenable to surgical resection, primary tumour in situ permitted (FOXFIRE); histologically confirmed CRC with liver-only or liver-dominant metastases (SIRFLOX); histologically confirmed adenocarcinoma of the colon or rectum with or without primary tumor in situ Unequivocal and measurable computed tomography (CT) evidence of liver metastases not treatable by surgical resection or local ablation with curative intent at time of trial entryChemotherapy-naïve for mCRC, but previous adjuvant systemic chemotherapy for primary CRC or neoadjuvant chemoradiotherapy to the pelvis more than 6 months before recruitment is permitted ; eligible for systemic chemotherapy as first-line treatment for metastatic CRC (FOXFIRE)Additional limited extrahepatic metastases in the lung or lymph nodes permitted ; patients are permitted to have limited extrahepatic disease (FOXFIRE)Age ≥18 yearsWorld Health Organization performance status of 0-1Life expectancy >3 monthsAdequate hematological, renal, and hepatic functionFemale patients must be postmenopausal or using adequate contraception if premenopausal, and male patients must be using an appropriate method of contraception if with a premenopausal partner (FOXFIRE); female patients must either be postmenopausal, sterile  or if sexually active using an acceptable method of contraception, and male patients must be surgically sterile or if sexually active and have a premenopausal partner must be using an acceptable method of contraception Suitable for all aspects of treatment determined by clinical assessment undertaken by investigatorWilling and able to provide written informed consentFOXFIRE-Global and SIRFLOX:All imaging evidence used as part of the screening process must be within 28 days prior to randomizationEvidence of ascites, cirrhosis, portal hypertension; tumour involvement of, or thrombosis leading to occlusion of the main portal veinPrevious radiotherapy delivered to upper abdomen or upper lumbar spine (FOXFIRE); previous radiotherapy delivered to the upper abdomen (SIRFLOX); previous radiotherapy delivered to the liver Nonmalignant disease that would render patient ineligible at the discretion of the investigatorDose-limiting toxicity associated with previous 5-fluorouracil or oxaliplatin chemotherapyPeripheral neuropathy higher than grade 1 Pregnant or breastfeedingPrevious chemotherapy for any malignancy; adjuvant chemotherapy for colorectal cancer is not an exclusion criterion provided that it was completed more than 6 months prior to entry into the study . Previous chemotherapy for metastatic colorectal cancer; adjuvant chemotherapy for colorectal cancer is not an exclusion criterion provided that it was completed more than 6 months prior to entry into the study (FOXFIRE)Concurrent or prior history of cancer other than adequately treated nonmelanoma skin cancer or carcinoma in situ of the cervix ; other active malignancy within last 5 years excluding colorectal cancer and other nonmelanoma skin cancers (FOXFIRE)SIRFLOX and FOXFIRE-Global:Allergy to nonionic contrast agentsFOXFIRE only:Liver metastases amenable to curative resection at time of study entryEquivocal, immeasurable, or nonevaluable liver metastasesUnequivocal evidence of bone metastasisAll patients received first-line chemotherapy for mCRC. In FOXFIRE, the chemotherapy received was oxaliplatin, 5-fluorouracil, and leucovorin/folic acid in the OxMdG regime . In SIRFIn all 3 trials, patients were assessed every 2 weeks during chemotherapy treatment. In SIRFLOX and FOXFIRE-Global, patients were assessed every 12 weeks during the postprogression follow-up period. FOXFIRE patients were assessed every 8 weeks following completion of treatment up to 18 months and then every 12 weeks thereafter. In all 3 trials, patients were followed until death or for a period of at least 2 years after randomization. Patients undergoing surgical resection after trial entry were also followed up until trial closure or until death.Screening and follow-up assessments included clinical assessment and laboratory analyses, recording concurrent medications, CT scan of the chest/abdomen/pelvis with or without magnetic resonance imaging, recording adverse events, assessment for resection or ablation, and questionnaire-based assessments of quality of life.The primary outcome of OS is the time from randomization to death from any cause, with patients still alive being censored at their last known follow-up date.The secondary outcomes are PFS at any site, liver-specific PFS, health-related quality of life (HRQoL), response rate, resection rate, and the safety profiles. PFS is defined as the time from randomization to radiological progression according to Response Evaluation Criteria In Solid Tumors (RECIST version 1.0) or death from any cause, whichever is sooner. Patients who are not observed to progress or die during the course of the trial will be censored at last known progression-free follow-up date. Patients who withdraw from study treatment prior to documented progression will be censored at the time they commence nonstudy treatment. Patients who become eligible for resection will be considered as still being on study until progression is documented or last follow-up. Scans and tumor response will be centrally reviewed in FOXFIRE and SIRFLOX.Liver-specific PFS is defined as the time from randomization to radiological progression in the liver (hepatic progression) according to RECIST version 1.0. Progression outside the liver (extrahepatic progression) and deaths prior to progression will be considered as competing risks for failure in the liver. Hepatic progression will be assumed to have occurred immediately before extrahepatic progression in patients who have identical hepatic and extrahepatic progression dates. Patients who withdraw from study treatment prior to any documented progression will be censored at the time they commence nonstudy treatment. Patients who become eligible for resection will be considered as still being on study until progression is documented or last follow-up.HRQoL will be assessed using the EuroQol 5 dimensions questionnaire (EQ-5D). The EQ-5D comprises the following 5 dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each of the 5 dimensions is scored, generating a profile. A single index score or utility value, representing population-derived preferences for different health states, will be attached to each profile.The response rate (objective response) is defined as the number of patients achieving a complete or partial response according to RECIST version 1.0 divided by the number of patients in each treatment arm. Early death by any cause and unknown responses will be included in the denominator. Response will be assessed over 2 time periods: within 12 months of randomization and over follow-up.The resection rate in each treatment arm is defined as the number of patients undergoing resection of their liver metastases divided by the number of patients randomized in each arm. Patients undergoing treatment are assessed by an experienced liver multidisciplinary team or equivalent for eligibility for resection at the discretion of the treating physician.The safety profile is assessed by the collection of adverse events (AEs) and serious adverse events (SAEs) at any time during treatment with grading using the NCI-CTCAE version 3.For the primary combined OS analysis, the FOXFIRE study protocol details a calculated 1075 patients required using a protocol-specified hazard ratio (HR) of 0.8, a 36-month accrual period, and 18-month minimum follow-up with 2-sided 5% significance, 80% power, and allowing for noncompliance. A total of 710 deaths are expected. The required sample size had been previously calculated to be 1022  but was The final analysis will be undertaken when a minimum of 710 deaths overall and 463 deaths in the liver-only subgroup have been observed in the pooled dataset and there has been a minimum follow-up of 2 years since the last patient was randomized into the 3 trials.The independent data and safety monitoring committee (IDSMC) consisted of the same representatives for the FOXFIRE, SIRFLOX, and FOXFIRE-Global clinical trials. Formal interim monitoring of the accumulating data was performed at regular intervals (approximately every 6 months) by the IDSMC for each trial separately. This information included results from other relevant trials but not the analysis of primary or secondary objectives or outcomes by treatment groups apart from the prespecified interim analyses. As part of the review, the IDSMC was asked to justify continued recruitment of further patients or further follow-up. The IDSMC advised on the frequency of future reviews of the data on the basis of accrual and event rates.The IDSMC reviewed the combined safety data from FOXFIRE and SIRFLOX at the interim analyses. The following planned interim analyses were undertaken using combined data from the FOXFIRE and SIRFLOX trials:Analysis of toxicity and safety: 8 months after at least 80 patients were randomized in total Analysis of toxicity and safety: 8 months after at least 300 patients were randomized in total The 3 clinical trials that constitute this MA were conducted in accordance with the Declaration of Helsinki and current Good Clinical Practice guidelines, and all participating centers obtained the relevant ethics committee approval before patient enrollment. FOXFIRE was approved by the National Research Ethics Service Committee South Central – Berkshire Research Ethics Committee reference 09/H0505/1 and sponsored by the University of Oxford. SIRFLOX and FOXFIRE-Global were approved by the relevant ethics committees for each center and sponsored by Sirtex Technology Pty Ltd.Summaries of baseline factors, including minimization factors, and percentages of missing data will be reported. Losses to follow-up will be reported for each trial and combined. Median follow-up time will be calculated using the reverse Kaplan-Meier method. Dose of trial-specific treatment delivered and treatment received subsequent-to-trial treatment will be described.All efficacy analyses will be performed on an intention-to-treat (ITT) basis. OS and PFS estimates will be obtained using Kaplan-Meier survival curves, unadjusted log-rank tests, and survival models. A 2-stage inverse-variance weighted IPD-MA will be performed for both OS and PFS, with the first stage consisting of trial-specific analyses to obtain efficacy estimates (HRs) and the second stage being a pooled analysis of the separate trial-specific HRs. As a sensitivity analysis, a 1-stage IPD-MA using regression models stratified by study will be performed to confirm the results obtained from the 2-stage IPD-MA. For each outcome, multivariable models will be used in the 1-stage IPD-MA to account for baseline covariates.Liver-specific PFS will be analyzed using cumulative incidence curves, Gray’s test, and competing risks regression. This analysis will be performed on the pooled dataset with models stratified by trial. For OS, PFS, and liver-specific PFS, the potential treatment benefit in those patients who present with disease confined to the liver will be investigated. This prespecified subgroup analysis will be performed on the pooled dataset. The analysis strategy will include calculating HR for the effect of treatment in the liver-only subgroup using survival models stratified by trial.A separate landmark analysis of OS will start at the 15-month time point (after randomization) and therefore exclude those who have died/withdrawn within 15 months of randomization. This time point has previously been considered of value in clinical trials of the treatment of mCRC . Based oThe EQ-5D data will be merged across all 3 trials and summary data prepared on the mean EQ-5D utility score in each trial arm by time period, with appropriate tests for difference. Resection rate and response rate will be analyzed using chi-square tests and odds ratios (ORs) for the individual trials and a 2-stage IPD-MA.Safety analyses will be performed on patients who received at least 1 dose of chemotherapy in either arm and on an as-treated basis (safety population). AEs experienced up to 28 days after the end of trial treatment or 7 months postrandomization, whichever was earlier, will be included. Descriptive summaries of the frequency and severity of AEs and the numbers of patients experiencing AEs of grade 3 or higher between treatment arms will be presented overall and by system organ class. The Medical Dictionary for Regulatory Activities version 16.1 is used to categorize the AEs. Univariate comparisons will be made.All hypothesis tests will be 2-sided. A significance level of .05 will be used to indicate statistical significance. No missing data imputation is intended. Missing days in dates may be appropriately imputed.Data from the final data lock will become available in January 2017. Data analysis will take place in 2017 with results being disseminated via peer-reviewed journals in 2017 and 2018.Optimizing outcomes of treatment in patients with nonresectable liver metastases was identified in an international consensus expert statement as a key clinical need to be addressed . This isThis prospective MA of 3 phase III studies will provide comprehensive evidence of the safety and potential efficacy of SIRT in the first-line setting for patients with liver metastases from mCRC. The results of the SIRFLOX trial published so far have suggested that the addition of SIRT to chemotherapy can improve liver-specific PFS . The numAlthough the SIRFLOX trial reported that liver-specific PFS was longer in the SIRT arm than in the control arm, the PFS at any site was similar between the SIRT arm and the control arm . This caIt is generally accepted that successful resection of liver metastases correlates with improved overall survival, particularly in the context of neoadjuvant or adjuvant chemotherapy ,22. SurgThe use of an IPD rather than an aggregate data approach to systematic review and MA of randomized controlled trials enables the standardization of outcomes across trials and detailed data checking, providing a more in-depth exploration and more robust MA results . The proThe results from the combined analysis of the SIRFLOX, FOXFIRE, and FOXFIRE-Global trials will provide valuable clinical information on the efficacy and toxicity profile of SIRT combined with chemotherapy as a first-line regimen for liver metastases from mCRC that will guide clinicians in the use of this technology to treat their patients. The IPD-MA will allow comparisons of less common AEs that would not be possible in a smaller population. Furthermore, this prospective MA provides sufficient power to determine whether SIRT provides a significant survival benefit for patients with metastases confined to the liver and no clinically detectable extrahepatic disease, an important research question among clinicians treating mCRC. When reported, the results of this combined analysis will define the use of SIRT in the treatment of mCRC.


There is increasing evidence that elevated body mass index (BMI) is associated with reduced survival for women with breast cancer. However, the underlying reasons remain unclear. We conducted a Mendelian randomization analysis to investigate a possible causal role of BMI in survival from breast cancer.We used individual-level data from six large breast cancer case-cohorts including a total of 36 210 individuals (2475 events) of European ancestry. We created a BMI genetic risk score (GRS) based on genotypes at 94 known BMI-associated genetic variants. Association between the BMI genetic score and breast cancer survival was analysed by Cox regression for each study separately. Study-specific hazard ratios were pooled using fixed-effect meta-analysis.P = 0.03). We observed no association for ER-negative cases .BMI genetic score was found to be associated with reduced breast cancer-specific survival for estrogen receptor (ER)-positive cases [hazard ratio (HR) = 1.11, per one-unit increment of GRS, 95% confidence interval (CI) 1.01–1.22, Our findings suggest a causal effect of increased BMI on reduced breast cancer survival for ER-positive breast cancer. There is no evidence of a causal effect of higher BMI on survival for ER-negative breast cancer cases. Observational studies have reported an association between elevated body mass index (BMI) and reduced survival for women with breast cancer. However, the causal nature of the association is unclear.We conducted a large Mendelian randomization analysis in order to examine a potential causal effect of BMI on breast cancer survival, using both individual genotype data and summary data.Our study provides evidence that the reported association between BMI and survival for estrogen receptor-positive breast cancer is likely to be causal.Breast cancer is the most common form of cancer for women worldwide.,Mendelian randomization (MR)The aim of this study was to utilize germline genotype data for genetic variants known to be associated with BMI, in a breast cancer case-cohort to evaluate the association between BMI and breast cancer survival in an unbiased way. There are three assumptions under which genetic variants provide valid instrumental variables for the effect of BMI on breast cancer survival: first, the genetic variants are associated with BMI; second, the variants are not associated with any confounder of the BMI-breast cancer survival association (pleiotropy); third, the variants are conditionally independent of the survival, given the BMI and confounders (exclusion restriction).IJE online). Genotypes for common variants across the genome were imputed using a reference panel from the 1000 Genomes Project (March 2012) for each dataset. All patients provided written informed consent, and each study was approved by the relevant institutional review board. Data on age at diagnosis, vital status, breast cancer-specific mortality, follow-up time, time between diagnosis and blood draw, lymph node status, histological grade, tumour size and estrogen receptor status were also available. In addition, some case-cohorts from the COGS study provided data on height and weight (self-reported) at date closest to diagnosis (cases) or study entry (controls) for 65 582 participants. BMI was calculated as weight in kilograms divided by height in metres squared (kg/m2).We included six datasets where a genotyping array providing genome-wide coverage of common genetic variation had been used to genotype multiple breast cancer case-cohorts in populations of European ancestry . A summary of these case-cohorts has been described in detail previously.2 of all 94 single nucleotide polymorphisms (SNPs) in the breast cancer dataset is greater than 0.4.The Genetic Investigation of Anthropometric Traits (GIANT) consortium involving over 300 000 individuals of European descent has reported 97 common variants associated with BMI, of which three were only associated with BMI for men.We verified the first assumption of Mendelian randomization by evaluating the association between BMI GRS and BMI in a set of control subjects from the COGS study. MR analysis was performed using Cox proportional hazard models, to evaluate the associations of the BMI genetic risk scores with breast cancer-specific mortality based on 36 210 cases with 2475 events over 170 504 person-years of follow-up. The date of diagnosis was used to calculate time-to-event with follow-up being censored at death, last follow-up or 10 years, whichever came sooner. As several studies include prevalent cases, the date of study entry was used to determine time under observation in order to adjust for the potential bias of prevalent cases in a prospectively recruiting study (left-truncation).We assessed the relationship between BMI GRS and breast cancer survival using summary statistics for the association of each BMI- associated SNP with survival, for each dataset. We used both an inverse-variance weighted method and a likelihood-based method2  increase in BMI and explained 1.6% of the BMI variance (F statistic = 450). Self-reported BMI was significantly associated with breast cancer survival for both ER-negative and ER-positive disease in the COGS data (We observed strong positive associations between the BMI GRS and observed BMI using a set of 28 190 controls from the COGS study. A one-unit increase in GRS corresponds to a 0.94 kg/mOGS data . Both asP = 0.03) per one-unit increment of the GRS (P = 0.93).We performed MR analysis for all available ER-negative and ER-positive breast cancer cases. The GRS was found to be associated with reduced breast cancer-specific survival for ER-positive cases with hazard ratio (HR) of 1.11 . The differences between the estimated associations with genetic score for ER-positive and ER-negative were not significant (P = 0.07). The association between BMI and breast cancer survival was also evaluated using standard inverse-variance weighted meta-analysis of summary statistics for the association of each BMI-associated SNP with survival. The results were similar to those based on individual-level data , suggesting no overall directional pleiotropy. A funnel plot of the minor allele frequency-corrected genetic associations with the BMI against the individual causal effect estimates for each SNP shows little evidence for asymmetry .We tested each GRS SNP for association with either node status or grade or tumour size or stage. Sixteen of the BMI SNPs were associated with one or more of these variables. We then repeated the individual data MR analysis using a GRS-78 that excluded these SNPs. The magnitudes of the associations with ER-positive breast cancer were similar to those for the results based on all the BMI SNPs . However, there was no association between smoking and prognosis . It is unlikely that the association between smoking behaviour and BMI can explain the association between BMI GRS and prognosis.We explored a potential complex relationship between smoking behaviour, BMI and prognosis by investigating the association between BMI GRS and smoking behaviour and between smoking behaviour and prognosis. There was a very weak correlation between GRS and number of pack-years smoked , the genetic variants may be associated with these confounders for the subpopulation of breast cancer patients. This is due to conditioning on a collider: if BMI is a causal risk factor for breast cancer risk, then conditioning on breast cancer risk  means that all common causes of breast cancer risk (including the genetic variants and confounders) are conditionally associated. In simple terms, even if genetic variants are distributed randomly in the population as a whole, they are not necessary randomly distributed in the ascertained population of breast cancer patients. This may lead to bias in the analysis, although it is unclear how serious this bias might be. In order to evaluate the potential for collider bias, we performed a simulation study in which we simulated data on a genetic risk score and an exposure (BMI in our example) for 100 000 individuals. For each individual, we simulated whether that individual had a positive breast cancer diagnosis as a binomial random variable. For each individual with a positive breast cancer diagnosis, we simulated the time-to-event for breast cancer progression as an exponential random variable. The genetic risk score was simulated as a normally distributed random variable, as was the confounder (assumed unmeasured), and the independent error term. The probability of breast cancer diagnosis was modelled as a function of the exposure. This leads to the collider (selection) bias: individuals with a breast cancer diagnosis  will have higher average levels of the exposure and confounder than those not included. While collider bias was observed for extreme values of the effect of the risk factor on disease status, it was not observed for values that are in line with the effect of BMI on breast cancer diagnosis as observed in previous investigations. Hence, while we would be cautious not to generalize the result of this limited simulation study to other analysis contexts, in this case there seemed to be little potential for bias and type 1 error rate inflation to arise due to collider bias.While our results suggest a causal association between BMI and survival for women with ER-positive breast cancer, BMI is, in itself, a complex phenotype. It is conceivable that more specific phenotypes related to body fat composition and distribution might be better predictors of outcome. Untangling such complex relationships with survival will require data on the association between germline genetic variation and specific body fat composition and distribution phenotypes. Potential mechanisms underlying effects of obesity on breast cancer survival are mediators such as members of the insulin/insulin-like growth factor family, adipocytokines secreted from adipose tissue and inflammatory cytokines.Our study, based on data from multiple large-scale genetic association studies of breast cancer, provides evidence that the reported association between BMI and survival for ER-positive breast cancer is likely to be causal. This suggests that BMI reduction in overweight women with ER-positive breast cancer might improve clinical outcomes.IJE online.Supplementary DataClick here for additional data file.


The early warning system could have significant utility as a cost-effective solution for wildfire managers to prioritize the deployment of wildfire protection resources in wildfire-prone landscapes across boreal-dominated ecosystems of North America, Europe, and Russia using open access Earth observations.Spring represents the peak of human-caused wildfire events in populated boreal forests, resulting in catastrophic loss of property and human life. Human-caused wildfire risk is anticipated to increase in northern forests as fuels become drier, on average, under warming climate scenarios and as population density increases within formerly remote regions. We investigated springtime human-caused wildfire risk derived from satellite-observed vegetation greenness in the early part of the growing season, a period of increased ignition and wildfire spread potential from snow melt to vegetation green-up with the aim of developing an early warning wildfire risk system. The initial system was developed for 392,856 km In the boreal forests of North America it is anticipated that climate warming will result in increases in fuel availability and wildfire severity2 with concomitant increases in wildfire danger and wildfire season length3, particularly for the circumboreal forest4. The effects of increasing human activity such as urbanization and resource extraction within boreal forests remain poorly understood with respect to potential human-caused wildfire ignitions and wildfire risk6. Humans ignited approximately 57% of all wildfires in Alberta, accounting for 63% of annual area burned  between 1996 and 20147. More than two-thirds of human-caused wildfires in Alberta occur during what is recognized across Canada as the spring burning window8, a period of increased ignition and wildfire spread potential spanning the time between the disappearance of snow and vegetation green-up. The spring burning window is most pronounced in Alberta, which ends when understory and overstory vegetation produce fresh leaves that are high in moisture content, reducing the potential of wildfire ignition and spread8. Alberta is a model jurisdiction for investigating human-caused wildfire risk during the springtime due to the confluence of human ignition sources and a seasonal change in fuel moisture modulated by climate and leaf phenology. For these reasons, May is the most active wildfire month resulting in the highest monthly area burned in Alberta7, and springtime burning is common to circumboreal ecosystems across North America, Europe, and Russia11.The importance of changing wildfire regimes under a changing climate has become a major focus of research and management efforts globally13. Fine fuels with diameters smaller than 2.5 cm tend to respond rapidly to abrupt changes in weather conditions  due to a high surface area to volume ratio that increases rates of evapotranspiration compared with larger fuels14. Current models of fuel moisture, such as the Canadian Forest Fire Danger Rating System, integrate wildfire weather data with empirical knowledge of evapotranspiration rates for different forest fuel types to estimate daily fuel availability and potential wildfire behavior14. These models are ledgers of fuel moisture that capture moisture content of fine fuels and large coarse fuels including deep soils and duff, accounting for daily wetting and drying as well as seasonal drying factors15. The Canadian system has been broadly adopted around the world, including across Europe, however similar systems of wildfire danger rating exist such as the National Fire Danger Rating System in the United States16 and the MacArthur Forest Fire Danger Index in Australia17. Although these systems are widely used around the world to estimate wildfire danger and wildfire behavior, the weather-based inputs are not direct measurements of fuel moisture, and the Canadian system makes broad assumptions about the homogeneity of fuel types. As a result, there remains unaccounted spatial variability in the estimate of wildfire behavior within and between wildfires from these indirect fuel moisture models18.Previous research has shown that the moisture content in fine fuels is a critical risk factor of human-caused wildfire activity20. Satellite observations from the Moderate Resolution Imaging Spectroradiometer (MODIS) provide a unique opportunity to observe and monitor fuel moisture from the flush of new vegetation, since healthy leaf area has long been shown to have a strong relationship with spectral greenness indices and leaf moisture21. Since leaf area is a proxy for foliar fine fuel moisture8 and is readily measured directly from satellite-derived greenness indices, fewer assumptions are required about site-specific fuel types when assessing wildfire spread potential. Moreover, using cloud-free composites of MODIS imagery, surface reflectance observations are available nearly every eight days at a spatial resolution of 250 m for the entire planet22. Thus, the spectral, spatial, and temporal resolutions of the satellite observations are ideal for monitoring the spring burning window as expressed by vegetation greenness for large areas.Satellite observations of vegetation greenness have been successfully used to estimate wildfire danger codes and fuel moisture codes for northern boreal forestsIn this paper we propose that satellite-observed greenness trends will be useful for estimating the peak of the spring burning window for any location, thus forming an early warning system for forecasting human-caused wildfire activity, which has wide application in forested ecosystems beyond the focus site. To do so, we investigated the relationship between fine temporal changes in satellite-derived greenness, and the commencement of the spring burning window as quantified by human wildfire ignitions in Alberta’s Forest Protection Area, the forested areas where wildfires are actively managed using provincial resources. To facilitate the development of the early warning system, we provide an assessment of the model error (in days) as a function of day-of-year of observation acquisition and show ground conditions of modelled predictions using a ground network of phenology cameras. The developed relationships of the spring burning window can augment existing decision support systems by providing near real-time proxy information of fuel moisture to wildfire managers for allocating suppression resources and planning prescribed burns.2 of land and freshwater ecosystems23, of which approximately 42% is considered to be forested24. The provincial government actively manages a wildfire protection area totaling 392,856 km2 or approximately 59% of the province25. The wildfire protection area spans 16 natural sub-regions with varying continental and interior climates , which vary along the large latitude and elevation gradients that dominate the province.The province of Alberta comprises 661,848 kmtes Fig. . Growing7. The historical wildfire database contains wildfire ignitions observed from a variety of sources including lookout observers, forest officers, the public (310-FIRE), wildfire patrols, aircraft, wildfire crews, infrared scans, and satellite imagery. Attributes are recorded for each wildfire ignition including the estimated date of ignition, the cause of ignition, and wildfire size after being extinguished. Small wildfire ignitions (<0.1 ha) likely represented illegal campfires and were removed from the dataset. Ignitions caused by lightning or without sufficient information on the date of occurrence were also removed. The remaining human-caused wildfire ignitions between 2000 and 2014 totaled N = 5,325 and accounted for approximately 45% of the total area burned represented in the database and approximately 92% of human-caused area burned. The number of human-caused ignitions that occurred between February 26 (DOY 57) and July 15 (DOY 196) totaled n = 4,017 or approximately 75% of all human-caused wildfire ignitions across all calendar years.Wildfire ignitions for Alberta were retrieved from the historical wildfire database for the years 2000 to 201428. However, wildfire risk is known to vary according to ignition source and season29. For example, it has been postulated that deciduous leaf phenology in the western Canadian interior forest provides a major climate feedback on seasonal patterns of temperature and precipitation, with the possible conclusion that evapotranspiration from the leaf-on forest is at least partly responsible for the development of thunderstorms in the region30. Thus, lightning ignitions are not a suitable proxy for human-caused wildfire activity during leaf-off conditions, and local environmental conditions such as rapid springtime greening of vegetation, time since last snow cover , and human-caused wildfire ignitions also need to be considered. The timing of vegetation greening shifts inter-annually, however vegetation greening has been shown to be relatively stable in terms of amplitude31. By contrast, it is challenging to know a priori when and where humans ignite wildfires or what weather conditions will be prevalent beyond several weeks. Therefore, our approach was first to relate the timing of human-caused ignitions to vegetation green-up and then use the satellite remote sensing data to predict green-up dates as an indicator of peak springtime human-caused wildfire risk.Recent studies have used lightning ignition data to estimate wildfire risk during the wildfire season32. The probability of a human-caused wildfire day during a given year may be estimated as a function of a Poisson distribution, which approximates the binomial process, where the number of wildfire starts occurring on any given DOY for all available years 2000–2014 from the historical wildfire database is the rate parameter λ12.Human-caused wildfire risk was modelled by converting wildfire ignition counts to wildfire days, defined as any day of the calendar year when one or more human-caused wildfire ignitions are reported. A wildfire day is a meaningful concept to wildfire managers who require a prediction of whether a given location will require wildfire protection resources on a given day and provides a well-established, robust statistical random variable for binomial modelling22. The eight-day MODIS composites represented a compromise between obtaining sufficient observations to track vegetation phenology over a given season while minimizing data gaps caused by clouds and atmospheric conditions22. For every year, a total of 18 eight-day composite MODIS images were available. The MODIS images were downloaded directly from the United States Geological Survey22, converted to a 10 degree North American Datum 1983 Transverse Mercator projection, and mosaicked to cover the province of Alberta. Wildfire managers in Alberta use broad ecological natural sub-regions to manage wildfire protection resources, so we adopted those regions for summarizing trends of satellite-derived greenness across the wildfire protection area of the province26.The MODIS sensor onboard the Aqua and Terra satellites acquires imagery in the red and near infrared spectral wavelengths for most northern latitudes on a daily basis at 250 m spatial resolution. We retrieved eight-day surface reflectance image composites generated for the MOD09Q1 Version 6 product between February 26 (DOY 57) and July 12 (DOY 193) for years 2000 to 201633. We first excluded low-quality observations using the quality control data distributed with the MOD09Q1 product. The annual NDVI curves were averaged to derive a mean greenness normal for each DOY and then each season between 2000 and 2016 was screened for noise by converting the annual NDVI observations to a z-score. Observations were flagged as noise if they exceeded the one-sided upper tail critical value at α = 0.10. Assessing the upper tail ensured that observations with significantly lower greenness than expected from the 17-year average were flagged as noise since these observations were likely related to error in atmospheric correction procedure. For each year from 2000 to 2016, a one-dimensional cubic spline was fit34 to the seasonal NDVI values for each MODIS pixel to temporally interpolate the 8-day MODIS composites to daily estimated values of NDVI. Green-up was defined as the date when the temporally interpolated NDVI curve reached the mean of the minimum and maximum NDVI values for the season, also known as the half-maximum35. This procedure resulted in a spatially-explicit estimation of vegetation green-up across the region. A mask was applied to extract pixels occurring within the wildfire protection area of Alberta and green-up statistics were further summarized by natural sub-regions ons Fig. . Vegetati=0, … NDVIn} in a year, where i indexes the observation position in the year out of n = 18 total observations in any year. The mean absolute error (in days) between the observed and the forecasted green-up DOY from the cubic spline model was calculated for each pixel and for each satellite observation replaced sequentially in the season. This process was repeated for all years and days-of-year, up to and including July 4 (DOY 185).We tested the ability of the half-maximum greenness system to accurately forecast the peak spring burning window by simulating real-time availability of satellite observations throughout each annual season. This was achieved by sequentially replacing the 2000–2016 mean NDVI values with actual satellite observations for any given day-of-year and then recalculating the green-up date using the new greenness curve. The greenness curves that emerged from concatenating actual greenness observations with the mean greenness normal were then used to estimate the green-up date for the cumulative set of satellite observations {NDVIin situ in the Foothills, Subalpine, and Alpine natural sub-regions during the growing season between April and October in 200938. The cameras acquired five red-green-blue photos daily over an hour at noon local standard time to compensate for varying shadow and illumination effects in the scene. From these sets of images, we show some examples of the forest condition on or around the dates that we predict high probability of a wildfire day based on the half-maximum green-up model.A network of 16 phenological cameras were installed http://wildwildfire.alberta.ca/resources/historical-data/spatial-wildwildfire-data.aspxwildfirewildfirewildfirewildfire. The 8-day surface reflectance MODIS products (MOD09Q1) are publicly available from http://e4ftl01.cr.usgs.gov/MOLT/MOD09Q1.006/. All processed data and results including estimations of vegetation green-up and forecast simulations are available from the corresponding author upon reasonable request.Alberta wildfire ignitions are publicly available from Human-caused wildfire activity during the years 2000 and 2014 peaked in May Fig. . Days onVegetation green-up varied from year-to-year within each natural sub-region Fig. . The AlpSpringtime human-caused wildfire activity was concomitant with satellite-observed green-up. Human-caused wildfire ignitions had a wider range in the season compared with green-up in the wildfire protection area. The interquartile ranges for vegetation green-up and human-caused wildfire ignitions overlapped completely for all years, confirming that wildfire activity peaked shortly following vegetation green-up during the spring burning window Fig. . The medThe mean absolute error (days) for the forecasted green-up date decreased in all natural sub-regions as more observations were made available in the season Figs  and 6. BFrom phenological camera observations within the Foothills and Subalpine natural sub-regions, it is apparent that leaf-off conditions in mixedwood stands were concomitant with the timing of peak human-caused wildfire activity in early May Figs  and 8. I39. These activities have transformed historically remote boreal forests into anthropogenically-modified landscapes that pose increased risks for human-caused wildfire ignitions. As human uses of boreal forests increases in the near term, we can expect that the legacy of wildfire suppression from the beginning of the 20th century may exacerbate persistent wildfire hazards around settlements and critical infrastructures.Globally, northern boreal forests are seeing an increase in industrial activity, including the establishment of road networks, oil and gas development, timber harvesting, and mining40. Additionally, increased human-caused wildfire activity can be expected to increase social costs such as displacement from evacuations and increased smoke concentrations near settlements and over large areas41. The wildfire that devastated the town of Fort McMurray, Alberta, in May 2016 was the costliest natural disaster in Canadian history and the second costliest wildfire globally, resulting in an estimated C$3.58 billion insurable damages42 and the mandatory evacuation of approximately 88,000 residents43. Additionally, the proximity of the Fort McMurray wildfire to one of the largest oil mining operations globally resulted in an estimated loss of 800,000 barrels per day due to production stoppages44. Thus, an economical early warning system is critical for forecasting wildfire spread potential and mitigating these socioeconomic costs in the wildland-urban interface from human-caused wildfires45. With the rapid increase in wildfire suppression activities in recent decades, the wildfire statistics in Alberta demonstrate that the spring burning window remains the most dangerous part of the year with respect to loss of property and infrastructure40.The economic costs associated with wildfire suppression are recognized to be very high in the early months of the wildfire season and the variability of wildfire-fighting costs from year-to-year increases uncertainty for wildfire managers8. These vegetation conditions contribute to increased wildfire frequency, area burned, and wildfire size during springtime. Historical and contemporary wildfire records show that the spring burning window results in increased wildfire activity between May and June, which coincide with observable rapid changes in the vegetation phenology using remote sensing observations from this study. The increasing popularity and use of inexpensive visible spectrum digital cameras offers notable potential for the monitoring and measurement of phenological events such as spring green-up46. Repeat photography allows for very fine temporal sampling, often at daily or hourly intervals, for monitoring vegetation. By mounting these camera systems on towers, data can be acquired at an intermediate scale of observation, providing a link between field-based observation methods and satellite-derived estimates. In addition, the cameras provide a visual link between satellite-observed greenness over larger spatial scales to field-based measurements and observations from local experts. Our results demonstrate that the phenological camera network utilized in this study provides evidence of leaf-off conditions in mixedwood sites during peak human-caused wildfire activity in early May. To be effectively integrated into a near-realtime monitoring and forecasting system, however, these remote cameras would require additional investment in wireless technology for downlinking the imagery.The ability of wildfire ignitions to quickly spread in early spring is a well-known phenomenon attributed to a dip in foliar moisture and the drying of fine fuelsi.e., lower overall human-caused wildfire risk). The phenological camera observations demonstrated that green-up was reliably detected within mixedwood forests  and this could possibly degrade the accuracy of a forecast especially for the higher elevation forests.Some challenges remain for accurately forecasting vegetation green-up in higher elevation areas with shorter growing seasons and less pronounced deciduous vegetation. In Alberta, these areas account for only 24% of the wildfire protection area Fig.  and are sts Figs  and 8, uSimulation of the greenness curves from the end of winter to the end of vegetation green-up indicate that the vegetation green-up period was forecasted with 10-day accuracy for the majority of wildfire protected lands in Alberta by March 22 (DOY 81). This provides key evidence that vegetation greenness may be used as a reliable proxy for fine fuel moisture content. The eight-day composites limit the temporal precision available from the satellite data and introduce some uncertainty in the forecast. There is a fundamental trade-off between acquiring cloud-free satellite observations from MODIS and having sufficient temporal resolution to undertake the forecast on a near-weekly basis. Although in some cases a wildfire start date may be estimated, we report the probability of a wildfire day in terms of the 8-day MODIS intervals (Fig.


Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed. Potyvirus, family Potyviridae  and probe-based [TM]) and by the standard grow-out test (GO). While all 30 tubers tested positive for PVY by the GO test, 26 tubers tested positive by RT-LAMP while only 19 tubers tested positive by both variants of real-time RT-PCR (Table , by the