HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence.

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

Znf385C mediates a novel p53-dependent transcriptional switch to control timing of facial bone formation.

Jaw formation involves an intricate series of molecular events, whereby a chondrogenic scaffold precedes osteogenesis. The mechanisms coupling timing of cartilage maturation to onset of bone differentiation are poorly understood, particularly for neural crest-derived bones of the head. Here we present a novel zebrafish gene/protein-trap Citrine-fusion line that reveals transient expression of the zinc-finger protein Znf385C in maturing chondrocytes of the jaw. Functional analysis shows that loss of Znf385C disrupts a distinct peak of p21(cip1/waf1) expression in the chondrocytes, as well as causes premature ossification of the zebrafish jaw. We find that Znf385C is expressed as two splice variants which act differentially to activate p21(cip1/waf1) and/or interact with p53 in subcellular compartments. Taken together, the results suggest that Znf385C acts as a developmental switch for p53 function that modulates cell cycle arrest of chondrocytes and regulates timing of jaw cartilage maturation and ossification.

Evolutionarily conserved role for SoxC genes in neural crest specification and neuronal differentiation.

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

Laminin ß1a controls distinct steps during the establishment of digestive organ laterality.

Visceral organs, including the liver and pancreas, adopt asymmetric positions to ensure proper function. Yet the molecular and cellular mechanisms controlling organ laterality are not well understood. We identified a mutation affecting zebrafish laminin ß1a (lamb1a) that disrupts left-right asymmetry of the liver and pancreas. In these mutants, the liver spans the midline and the ventral pancreatic bud remains split into bilateral structures. We show that lamb1a regulates asymmetric left-right gene expression in the lateral plate mesoderm (LPM). In particular, lamb1a functions in Kupffer's vesicle (KV), a ciliated organ analogous to the mouse node, to control the length and function of the KV cilia. Later during gut-looping stages, dynamic expression of Lamb1a is required for the bilayered organization and asymmetric migration of the LPM. Loss of Lamb1a function also results in aberrant protrusion of LPM cells into the gut. Collectively, our results provide cellular and molecular mechanisms by which extracellular matrix proteins regulate left-right organ morphogenesis.

Zebrafish stem/progenitor factor msi2b exhibits two phases of activity mediated by different splice variants.

The Musashi (Msi) family of RNA-binding proteins is important in stem and differentiating cells in many species. Here, we present a zebrafish gene/protein trap line gt(msi2b-citrine)(ct) (57) (a) that expresses a Citrine fusion protein with endogenous Msi2b. Our results reveal two phases of Msi2b expression: ubiquitous expression in progenitor cells in the early embryo and later, tissue-specific expression in differentiating cells in the olfactory organ, pineal gland, and subpopulations of neurons in the central nervous system (CNS). Interestingly, this division between early and late phases is paralleled by differential expression of msi2b alternative splicing products. Whereas the full-length and long variant v3 Msi2b predominate at early stages, the later expression of variants in differentiating tissues appears to be tissue specific. Using the gt(msi2b-citrine)(ct) (57) (a), we characterized tissue-specific expression of Msi2b with cellular resolution in subsets of differentiating cells in the olfactory organ, pineal gland, CNS, and ventral neural tube. By performing transcription activator-like effectors nuclease-mediated biallelic genome editing or morpholino knockdown of Msi2b in zebrafish, our results show that early inactivation of Msi2b results in severe embryonic defects including hypertrophy of the ventricles and shortening of the body, consistent with an important role in cell proliferation and survival. Moreover, specific inactivation of Msi2b full-length indicates that this species is essential for the early role of Msi2b. This line provides a valuable tool both for live imaging of the endogenous Msi2b at subcellular resolution and manipulation of Msi2b-expressing cells.

Anconeus motor unit number estimates using decomposition-based quantitative electromyography.

INTRODUCTION: Motor unit number estimates (MUNEs) provide important information in health, aging, and disease, and can be determined using decomposition-enhanced spike-triggered averaging (DE-STA). Discrimination of surface-detected motor unit potentials (S-MUPs) has been limited to contractile forces of ∽30% maximum voluntary contraction (MVC), which is insufficient to recruit a representative sample of the entire MU pool in most muscles. Unique features of the anconeus may permit MUNEs at high muscle activation levels. METHODS: In 10 men (25 ± 3 years), anconeus MUNEs were performed using DE-STA at 10%, 30%, and 50% root-mean-square of MVC (RMS(MVC)). RESULTS: The mean compound muscle action potential of the anconeus was ∽6 mV, and average S-MUP amplitudes were ∽100 µV, 145 µV, and 235 µV at 10%, 30%, and 50% RMS(MVC), resulting in low average MUNEs of 58, 38, and 25, respectively. CONCLUSIONS: Elbow extensor force-EMG relationships suggest full recruitment of the anconeus MU pool at 50% RMS(MVC), thus providing a representative sample for MUNE.

The role of tissue diagnosis prior to neoadjuvant chemoradiotherapy for locally advanced rectal cancer.

AIM: Neoadjuvant chemoradiotherapy is the standard of care for locally advanced rectal cancer, with diagnostic work-up routinely including a biopsy confirming invasive carcinoma. For the occasional patient where initial biopsies reveal only dysplasia, or even normal epithelium, repeat biopsy is currently advised, but this may delay therapy and repeat biopsy has potential adverse effects. The study aimed to determine, in the setting of clinical findings and imaging demonstrating locally advanced rectal cancer, whether the absence of a tissue diagnosis prior to commencing chemoradiation compromises patient outcome. METHOD: A review was conducted of our database, including comprehensive treatment and outcome details, in which consecutive patients with colorectal cancer have been enrolled since 1997 at a single institution. All records for patients who received neoadjuvant chemoradiotherapy for locally advanced rectal cancer were reviewed to identify patients for whom treatment was initiated before a tissue diagnosis was obtained, and to assess any consequences of this. RESULTS: Of 254 patients who had received neoadjuvant treatment for rectal cancer, 16 (6.3%) were found to have had neoadjuvant therapy without a tissue diagnosis of invasive cancer. Compared with cases where a tissue diagnosis had been obtained, median age (59 vs 63 years, P = 0.497), sex (75% vs 71.3% male, P = 0.955) and tumour location (56.3% vs 73.5% < 8 cm, P = 0.230) were similar. Of these, 14 (87.5%) had adenocarcinoma identified on histopathology review of the surgical specimen. Three patients were considered to have had complete pathological responses with mucin lakes within the muscularis propria (n = 2) or lymph nodes (n = 1) or fibrosis (n = 3). One of these had no mucin evident and only fibrosis; thus final pathological proof of invasive cancer was present in 15 (93.5%) patients. There were no local recurrences, but three of the 16 (18.8%) cases developed distant recurrence. CONCLUSION: For the small number of cases without a confirmatory tissue diagnosis before chemoradiation, no adverse consequences were identified. In particular the initial diagnosis was confirmed in 15 out of 16 cases following pathological examination of the operative specimen. We would suggest that, where clinical and radiological features support a diagnosis of locally advanced rectal cancer, proceeding directly to neoadjuvant chemoradiotherapy in the absence of a biopsy demonstrating invasive cancer may not be unreasonable, particularly where repeat biopsy would delay the commencement of treatment.

High-contrast, synchronous volumetric imaging with selective volume illumination microscopy.

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

Pre-processing visualization of hyperspectral fluorescent data with Spectrally Encoded Enhanced Representations.

Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER's enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis.

Angioplasty in critical limb ischaemia: one-year limb salvage results.

INTRODUCTION: Lower extremity amputation prevention (LEAP) is an ongoing programme in our institution aimed at limb salvage in patients with critical limb ischaemia (CLI). Patients in the LEAP programme with reconstructible anatomy on initial Doppler imaging received either bypass surgery or percutaneous transluminal balloon angioplasty (PTA). We describe the 1-year limb salvage rates in 46 consecutive patients with CLI who received PTA in 2005. CLINICAL PICTURE: A total of 46 patients, 28 women and 18 men, between the ages of 40 and 91 years old (mean age, 70.8) received PTA in 2005. The most common presenting symptom was rest pain (n = 23), followed by pre-existing gangrene (n = 20), non-healing ulcer (n = 17) and cellulitis (n = 8). The majority of the patients (57%) had 3 to 4 risk factors. Diabetes mellitus (91%) and hypertension (80%) were the 2 most common risk factors. The patients were kept under surveillance for periods ranging from 12 to 21 months with a mean of 13.3 months, both clinically and with haemodynamic measurements [ankle-brachial index (ABI), toe pressure (TP) and digital-brachial index (DBI)]. TREATMENT: The aim of PTA is to achieve straight-line flow from the abdominal aorta down to either a patent dorsalis pedis or plantar arch with limb salvage as the ultimate goal. The patterns of the treated segments were as follows: aorto-iliac occlusions (n = 3), pure infrapopliteal disease (n = 5), femoropopliteal disease with at least 1 good infrapopliteal run-off vessel (n = 16) and combined femoropopliteal and infrapopliteal disease (n = 25). Technical success was achieved in 89% of patients (41 out of 46 patients). The most common cause of technical failure is the inability to cross long chronic total occlusions. OUTCOME: Paired T test was performed and showed statistically significant improvement in haemodynamic markers within the technically successful group. This included increase in the mean ABI from 0.62 (preangioplasty) to 0.91 (Day 1 post-angioplasty), an increase of 0.29 [95% confidence interval (95% CI), 0.1953 to 0.3875; P <0.001]. One year post-angioplasty, the mean ABI was 0.84, an increase of 0.22 (95% CI 0.1512 to 0.3121; P <0.001). There was also significant increase in the mean DBI of 0.17 from 0.23 to 0.41 (pre-angioplasty versus Day 1 post angioplasty - 95% CI of 0.1006 to 0.2433; P <0.001). In addition, significant increase in the mean TP of 28.2 mmHg from 36.8 to 63.2 mmHg (pre-angioplasty versus Day 1 post angioplasty - 95% CI, 18.493 to 37.939; P <0.001) was also noted. Of the 23 patients who presented with rest pain, total abolishment of symptoms was achieved in 21 patients (91%). Healing of pre-existing gangrene was attained in 15 patients (66%). Five patients subsequently received minor amputation for pre-existing gangrene. Clinical improvement in all the patients who presented with non-healing ulcers (n = 17) and cellulitis (n = 8) was attained. More importantly, all healed ulcers remained healed throughout the study period. The limb salvage rates were 93% at 1 month, 87% at 3 months, 82% at 6 months and 78% at 1 year. CONCLUSION: Angioplasty is a safe and effective limb salvage method in patients with CLI and has a high 1-year limb salvage rate.

A comparison study of the volar wrist cock-up splint and ulnar gutter splint in carpal tunnel syndrome.

The objective of the study was to compare the effects of the volar wrist cock-up splint and the ulnar gutter splint on three dependent measures: level of comfort, pain relief and hand function, in clients with early symptoms of carpal tunnel syndrome. Four clients with initial onset of carpal tunnel syndrome symptoms participated in a study with alternating treatment design that compared the effects of the two splints. Comfort and pain were measured with perceived analogue scales and hand function was assessed using six sub-tests of the Jebsen Hand Function test. Evaluations were done every 2 weeks concurrent with the alternation of the splints for a period of 8 weeks. Descriptive analysis was used to identify differences in the dependent measures. Three participants showed improved performance in hand function tests with reduced time taken in tests following use of splints. Three participants showed reduced pain after splint use. One participant did not show changes with time taken in hand function tests or with level of pain. The study showed clinical evidence that both the splints contributed to alleviation of carpal tunnel syndrome symptoms. It is imperative that more efficacy studies be done to gain more confidence with the results of this study.

Angioplasty in critical limb ischaemia: one-year limb salvage results

<p><b>INTRODUCTION</b>Lower extremity amputation prevention (LEAP) is an ongoing programme in our institution aimed at limb salvage in patients with critical limb ischaemia (CLI). Patients in the LEAP programme with reconstructible anatomy on initial Doppler imaging received either bypass surgery or percutaneous transluminal balloon angioplasty (PTA). We describe the 1-year limb salvage rates in 46 consecutive patients with CLI who received PTA in 2005.</p><p><b>CLINICAL PICTURE</b>A total of 46 patients, 28 women and 18 men, between the ages of 40 and 91 years old (mean age, 70.8) received PTA in 2005. The most common presenting symptom was rest pain (n = 23), followed by pre-existing gangrene (n = 20), non-healing ulcer (n = 17) and cellulitis (n = 8). The majority of the patients (57%) had 3 to 4 risk factors. Diabetes mellitus (91%) and hypertension (80%) were the 2 most common risk factors. The patients were kept under surveillance for periods ranging from 12 to 21 months with a mean of 13.3 months, both clinically and with haemodynamic measurements [ankle-brachial index (ABI), toe pressure (TP) and digital-brachial index (DBI)].</p><p><b>TREATMENT</b>The aim of PTA is to achieve straight-line flow from the abdominal aorta down to either a patent dorsalis pedis or plantar arch with limb salvage as the ultimate goal. The patterns of the treated segments were as follows: aorto-iliac occlusions (n = 3), pure infrapopliteal disease (n = 5), femoropopliteal disease with at least 1 good infrapopliteal run-off vessel (n = 16) and combined femoropopliteal and infrapopliteal disease (n = 25). Technical success was achieved in 89% of patients (41 out of 46 patients). The most common cause of technical failure is the inability to cross long chronic total occlusions.</p><p><b>OUTCOME</b>Paired T test was performed and showed statistically significant improvement in haemodynamic markers within the technically successful group. This included increase in the mean ABI from 0.62 (preangioplasty) to 0.91 (Day 1 post-angioplasty), an increase of 0.29 [95% confidence interval (95% CI), 0.1953 to 0.3875; P <0.001]. One year post-angioplasty, the mean ABI was 0.84, an increase of 0.22 (95% CI 0.1512 to 0.3121; P <0.001). There was also significant increase in the mean DBI of 0.17 from 0.23 to 0.41 (pre-angioplasty versus Day 1 post angioplasty - 95% CI of 0.1006 to 0.2433; P <0.001). In addition, significant increase in the mean TP of 28.2 mmHg from 36.8 to 63.2 mmHg (pre-angioplasty versus Day 1 post angioplasty - 95% CI, 18.493 to 37.939; P <0.001) was also noted. Of the 23 patients who presented with rest pain, total abolishment of symptoms was achieved in 21 patients (91%). Healing of pre-existing gangrene was attained in 15 patients (66%). Five patients subsequently received minor amputation for pre-existing gangrene. Clinical improvement in all the patients who presented with non-healing ulcers (n = 17) and cellulitis (n = 8) was attained. More importantly, all healed ulcers remained healed throughout the study period. The limb salvage rates were 93% at 1 month, 87% at 3 months, 82% at 6 months and 78% at 1 year.</p><p><b>CONCLUSION</b>Angioplasty is a safe and effective limb salvage method in patients with CLI and has a high 1-year limb salvage rate.</p>