Renal Pelvic Urothelial Carcinoma With Invasion Into Renal Medulla Can Be Redefined as pT2 to Improve Correlation With Survival.

According to the American Joint Cancer Committee, pT3 renal pelvic carcinoma is defined as tumor invading the renal parenchyma and/or peripelvic fat and is the largest pT category, with notable survival heterogeneity. Anatomical landmarks within the renal pelvis can be difficult to discern. Using glomeruli as a boundary to differentiate renal medulla invasion from renal cortex invasion, this study aimed to compare patient survival of pT3 renal pelvic urothelial carcinoma on the basis of the extent of renal parenchyma invasion and, thereafter, determine whether redefining pT2 and pT3 improves pT correlation with survival. Cases with primary renal pelvic urothelial carcinoma were identified through a review of pathology reports from nephroureterectomies completed at our institution from 2010 to 2019 (n = 145). Tumors were stratified by pT, pN, lymphovascular invasion, and invasion of the renal medulla versus invasion of the renal cortex and/or peripelvic fat. Overall survival between groups was compared using Kaplan-Meier survival models and Cox regression multivariate analysis. pT2 and pT3 tumors had similar 5-year overall survival, with multivariate analysis demonstrating an overlap between hazard ratios (HRs) for pT2 (HR, 2.20; 95% CI, 0.70-6.95) and pT3 (HR, 3.15; 95% CI, 1.63-6.09). pT3 tumors with peripelvic fat and/or renal cortex invasion had a 3.25-fold worse prognosis than pT3 tumors with renal medulla invasion alone. Furthermore, pT2 and pT3 tumors with only renal medulla invasion had similar overall survival, whereas pT3 tumors with peripelvic fat and/or renal cortex invasion had a worse prognosis (P = .00036). Reclassifying pT3 tumors with only renal medulla invasion as pT2 yielded greater separation between survival curves and HR. Thus, we recommend redefining pT2 renal pelvic carcinoma to include renal medulla invasion and restricting pT3 to peripelvic fat and/or renal cortex invasion to improve the prognostic accuracy of pT classification.

Cutaneous Atypical Fibroxanthoma With Osteoclast-Like Giant Cell: A Rare but Diagnostic Pitfall.

BACKGROUND: Atypical fibroxanthoma (AFX) is a dermal-based, low-grade neoplasm with no specific lineage of differentiation. The occurrence of AFX with osteoclast-like giant cells is exceptionally rare. Less than 20 cases have been reported in the literature. CASE PRESENTATION: A 77-year-old man with a medical history of multiple basal and squamous cell carcinomas of the skin, presented with a progressively growing erythematous nodule on the sun-damaged right central parietal scalp. A shave biopsy showed a dermal spindle cell proliferation accompanied by numerous osteoclast-like multinucleated giant cells and predominant atypical mitotic figures. The immunohistochemical staining showed a diffuse positive staining for CD68 and SMA, patchy staining for CD10, and negative staining for SOX-10, pan-cytokeratin, CK5/6, S100, CD34, and desmin. The tumor was completely excised with negative margins. A subsequent follow-up over a period of 13 months showed no recurrence. CONCLUSION: Distinguishing AFX with osteoclast-like giant cells from both malignant and benign skin lesions with osteoclast-like giant cells is crucial. Although AFX tumors display worrisome malignant histologic features, most cases have a favorable prognosis with a local recurrence rate below 5% and exceedingly rare metastasis.

A Novel Macrophage-Activating Gel Improves Healing and Skin Quality After CO2 Laser Resurfacing of the Chest.

BACKGROUND: After laser resurfacing, it is imperative that an appropriate postoperative regimen is followed for optimal wound healing. There is currently no consensus about which agents should be used. OBJECTIVE: To evaluate the safety and efficacy of a novel macrophage-activating gel in a Phase 2B trial to be used after fractionated ablative laser resurfacing of the chest. MATERIALS AND METHODS: Forty-two adults who received fractionated CO2 laser resurfacing of the chest were randomized (active or placebo) for 5 consecutive days after procedure. Skin quality at baseline and follow-up was assessed by a blinded evaluator using the Fitzpatrick-Goldman Wrinkle Scale. Subject satisfaction with skin healing and quality was also assessed. RESULTS: At 28 days according to the Fitzpatrick-Goldman Wrinkle Scale, 85% of subjects achieved an improvement of at least 33% for the active group versus 50% in the placebo group (absolute difference 35%; p = .04). Similarly, 75% of subjects achieved an improvement score of at least 33% in elastosis in the active group versus 35% in the placebo group at 28 days (40% absolute difference; p = .011). CONCLUSION: This study confirms the potent effects of the novel macrophage-activating gel for optimization of skin healing and quality after laser resurfacing of the chest.

Serious Adverse Events With Injectable Fillers: Retrospective Analysis of 7,659 Patient Outcomes.

BACKGROUND: In total, 2.7 million injectable filler treatments were performed in 2019 in the United States. Although generally considered to be a safe treatment modality, adverse events may occur in rare situations. OBJECTIVE: Analyze serious adverse events from injectable filler treatments, including infections, cutaneous necrosis, blindness, or delayed-onset nodule formation, spanning 11 years for 3 board-certified dermatologists and review their incidence, management, and outcomes. MATERIALS AND METHODS: A retrospective analysis was performed of injectable filler treatments spanning 11 years at a multipractitioner outpatient clinic. Serious adverse events were identified, and treatment measures were documented. A literature search was performed to determine recent trends and outcomes for comparison. RESULTS: Between January 2009 and August 2020, 18,013 mL of injectable filler was administered to 7,659 patients. Of the 18,013 mL administered, 74.1% comprised hyaluronic acid derivatives, 19.19% poly-l-lactic acid, and 6.71% calcium hydroxylapatite. Four serious adverse events were identified. Three events were delayed-onset skin nodule formation. One adverse event was related to vascular compromise and subsequent cutaneous necrosis. After appropriate treatment, all adverse events resolved without significant long-term sequelae. CONCLUSION: Serious adverse events associated with injectable fillers, when performed by board-certified dermatologists, are extremely rare and can be successfully managed with appropriate treatment.

A Randomized, Evaluator-Blind, Split-Face Study Evaluating the Safety and Efficacy of Calcium Hydroxylapatite for Jawline Augmentation.

INTRODUCTION: Jawline augmentation with calcium hydroxylapatite has not yet been evaluated in a prospective study with a split-face design. This study aims to perform the first randomized controlled, split-face study on the efficacy and safety of calcium hydroxylapatite for jawline augmentation using the needle and cannula technique. OBJECTIVE: To perform the first randomized controlled, split-face study on the efficacy and safety of calcium hydroxylapatite for jawline augmentation using the needle and cannula technique. MATERIALS AND METHODS: This is a single-site, randomized, evaluator-blind trial enrolling a total of 10 healthy subjects with at least Grade 1 (mild) on a 4-point Jawline Scale. One side of the face was randomized to receive 1 to 2 syringes of calcium hydroxylapatite with lidocaine (total of 3 mL) for correction of wrinkles and folds along the jawline using both the cannula and needle method, and a balancing treatment will be performed 1 month later. Blinded investigator and subject evaluations will be performed immediately after treatment and at the 30-, 60-, and 90-day visits. RESULTS: Ten subjects were enrolled and completed the trial. There was a improvement in the degree of wrinkling and skin sagging in the 4-point Jawline Scale, with an average of a 1.3-point improvement in the scale on the day of treatment and at the Day 30 visit, which remained improved greater than baseline after 3 months as graded by blinded investigators. The Clinician Global Aesthetic Improvement Score for the treated side versus control, as assessed by blinded investigators, demonstrated a improvement with a 2.3-point improvement on the 5-point scale, and by the final visit on Day 90, most patients had a much improved appearance from baseline. CONCLUSION: This study demonstrates that calcium hydroxylapatite is effective and safe for restoration and augmentation of the jawline using the unique needle and cannula technique.

Novel Application of the 730 and 785 nm Picosecond Titanium Sapphire Lasers for the Treatment of Nevus of Ota.

BACKGROUND AND OBJECTIVES: Nevus of Ota is a benign dermal melanocytosis that may pose significant psychosocial distress to patients. Q-switched nanosecond lasers have traditionally been considered the first-line treatment but pain, bleeding, and postinflammatory pigmentary alteration are common adverse effects. Picosecond devices have been increasingly used to treat nevus of Ota with promising results. We present two cases demonstrating novel applications of the 730 and 785 nm picosecond titanium sapphire lasers for the treatment of nevus of Ota in two patients with types III and IV skin. STUDY DESIGN/MATERIALS AND METHODS: A 730 and 785 nm picosecond titanium sapphire laser with pulse durations of 250 and 300 picoseconds, respectively, were used to treat two cases of nevus of Ota. Four to seven treatment sessions were conducted at monthly intervals, and follow-up evaluation was performed 1-3 months following the final treatment session. RESULTS: Both cases demonstrated greater than 75% clearance following treatment. There were no adverse events or pigmentary alteration noted as a result of picosecond titanium sapphire laser treatment. CONCLUSIONS: The 730 and 785 nm picosecond titanium sapphire lasers are safe and effective for the treatment of nevus of Ota. Lasers Surg. Med. 00:00-00, 2021. © 2021 Wiley Periodicals LLC.

Safety and Efficacy of a Novel 730 nm Picosecond Titanium Sapphire Laser for the Treatment of Benign Pigmented Lesions.

BACKGROUND AND OBJECTIVES: Picosecond lasers in dermatology were originally focused on optimizing the removal of unwanted tattoos. Subsequent advances in this technology have expanded its clinical indications to include treatment of benign pigmented lesions, photodamage, melasma, and scar revision. In this retrospective review, we evaluate a novel 730 nm picosecond titanium sapphire laser in treating benign pigmented lesions. STUDY DESIGN/MATERIALS AND METHODS: This is a retrospective review of all patients who presented to our institution between December 2019 and March 2020 for treatment of their benign pigmented lesions with a 730 nm picosecond titanium sapphire laser. All Fitzpatrick skin types (I-VI) were included. Absolute and relative evaluations were conducted by two blinded board-certified dermatologists using high-resolution photographic images. RESULTS: Twenty-two of 64 patients satisfied inclusion and exclusion criteria. Patients received 1.1 ± 0.3 treatment sessions. The absolute average pigmentation score prior to treatment was 2.04 ± 0.7 versus 1.39 ± 0.6 after treatment (P < 0.05). Improvement in pigmentation was observed in 86% of the patients, while 3% had no improvement and 11% had worsening of pigmentation. No other adverse events were observed. Downtime consisted of 1-2 days of mild edema and erythema followed by 3-5 days of mild pigment darkening and superficial crust. CONCLUSION: The novel 730 nm picosecond titanium sapphire laser is a safe and effective treatment for benign pigmented lesions. Future prospective randomized control studies would be beneficial to further clarify its role in the treatment of benign pigmentation. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

A Systematic Review of Picosecond Laser in Dermatology: Evidence and Recommendations.

BACKGROUND AND OBJECTIVES: The use of picosecond laser in dermatology was originally focused on optimizing the removal of unwanted tattoos. Subsequent advances in this technology have broadened its clinical indications to include treatment of benign pigmented lesions, photodamage, melasma, and scar revision. In this systematic review, evidence-based recommendations are developed for the use of picosecond laser in dermatology. STUDY DESIGN/MATERIALS AND METHODS: A comprehensive search of the English language literature was performed up to and including November 2019. Relevant citations were individually evaluated, synthesized, and categorized based on the Level of Evidence. With the addition of the authors' combined clinical experience, clinical recommendations were developed. RESULTS: After application of inclusion and exclusion criteria, a total of 77 unique studies were evaluated. Treatment of benign pigmented lesions was associated with level I-IV evidence; rejuvenation was associated with level II evidence; melasma was associated with level II evidence; scar revision was associated with level II-III evidence; tattoo removal was associated with level I evidence. CONCLUSION: Picosecond laser is a safe and effective treatment modality for an increasing range of dermatologic indications. Further development of this technology is warranted. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

A Randomized, Split-Face, Double-Blind Comparison Trial Between Fractionated Frequency-Doubled 1064/532 nm Picosecond Nd:YAG Laser and Fractionated 1927 nm Thulium Fiber Laser for Facial Photorejuvenation.

BACKGROUND AND OBJECTIVE: Fractionated lasers are a popular therapeutic option for facial photorejuvenation. In this study, we compare the safety, tolerability, and efficacy of a fractionated frequency-doubled 1064/532 nm picosecond Nd:YAG fractionated picosecond laser (FPL) versus a fractionated 1927 nm thulium fiber laser (TFL) for facial rejuvenation. STUDY DESIGN/MATERIALS AND METHODS: This was a double-blind, randomized, split-face comparison study involving 20 subjects. Facial halves were randomized to receive either FPL or TFL treatment. Three treatments were delivered at 1-month intervals. Subjects were followed up for 1, 3, and 6 months post-final treatment session and evaluated by blinded, non-treating investigators for dyspigmentation, erythema, keratosis, texture, and rhytids on a standardized scale. Subjects also recorded a quantitative daily diary rating healing progress for 14 days after every treatment session. RESULTS: Statistically significant improvements in elastosis, erythema, keratosis, dyschromia, and skin texture were noted in both treatment groups. There were no significant differences detected in clinical efficacy between the two groups. Subject daily dairies revealed statistically significant differences in tolerability during the immediate 14-day post-operative recovery period. The facial half treated with FPL displayed significantly less redness on days 3 and 4; significantly less swelling on day 5; significantly less crusting on days 1 through 9; significantly less peeling on days 3 through 9; and significantly less itch on days 4 and 7. There were no unexpected adverse effects observed. CONCLUSION: Both FPL and TFL are safe and effective treatment options for facial rejuvenation. FPL may be associated with significantly less downtime. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

Intense Pulsed Light: A Methodical Approach to Understanding Clinical Endpoints.

BACKGROUND: Intense Pulsed Light (IPL) is a non-coherent polychromatic broadband filtered flashlamp that emits light in the spectrum of approximately 400–1200 nm. Its effects on photorejuvenation are well documented. The goal of this study is to help practitioners better conceptualize and fine tune IPL device settings in order to produce the most effective and safest clinical outcome. MATERIALS/METHODS: This was a prospective study testing several filters (515 nm; 560 nm; 590 nm and 530–650; 900–1200 nm vascular filter), fluences, pulse durations, and pulse numbers (ie, multiple sequence pulsing or MSP) with a new IPL system. RESULTS: Post-procedure erythema response was more pronounced with increasing fluence, decreasing wavelength, fewer pulses and shorter pulse duration. The exception was the 515 nm filter with regard to pulse duration, which was observed to have a more pronounced response with longer pulse durations. The overall clinical outcome at the 4-week follow-up visit demonstrated greatest improvement in erythema and pigmentation using the 515 nm filter on a Fitzpatrick Skin Type III individual. CONCLUSION: Greatest clinical endpoint response at 4-week follow-up was observed with more robust initial responses. This was most apparent at higher fluence levels and fewer pulse counts. However, when the IPL is pushed to aggressive parameters, there is risk of hypopigmentation and hair loss as seen in this case study. Skin type is an important consideration when using IPL and MSP adds to its safety profile. J Drugs Dermatol. 2021;20(2):203-207. doi:10.36849/JDD.5638.

AbobotulinumtoxinA Treatment of Glabellar Lines Using a New Reconstitution and Injection Volume: Randomized, Placebo-Controlled Data.

BACKGROUND: Increasing the reconstitution and injection volumes of abobotulinumtoxinA (aboBoNT-A) could provide more options for aesthetic healthcare professionals. OBJECTIVE: To evaluate efficacy and safety of aboBoNT-A treatment of moderate-to-severe glabellar lines (GL) versus placebo, using a new reconstitution and injection volume. Methods & Materials: In this 6-month, Phase III, randomized, double-blind study, subjects 18-64 years were administered aboBoNT-A 50 U (N=224) or placebo (N=77), as five 0.1-mL-injections (10 U) in the glabellar region following reconstitution of a 300-U-vial in 3 mL. Assessments included time to onset of effect, investigator- (ILA) and subject- (SSA) assessed GL severity, subject satisfaction, aesthetic improvement and safety. The primary endpoint was composite 2-grade response at month 1 (a GL severity of none-or-mild at maximum frown and ≥2-grade improvement from baseline concurrently on both ILA and SSA). RESULTS: Median time to onset was 2 days, 34% of subjects reporting effect on day 1. At month 1, the composite 2-grade responder rate was 65.8% for aboBoNT-A versus 0% for placebo, P<0.001, 91–92% had none-or-mild GL severity, and 95–100% had a ≥1-grade GL severity improvement. A ≥1-grade improvement was sustained in 46-56% of aboBoNT-A-treated subjects up to 6 months (P<0.001 vs placebo). Aesthetic improvement and subject satisfaction were high throughout 6 months and aboBoNT-A treatment was well tolerated. CONCLUSION: Safety and efficacy of GL treatment using 0.1 mL (10 U) aboBoNT-A per injection site were demonstrated, with rapid onset and up to 6 months’ duration of effect. Severity improvement was accompanied by sustained aesthetic improvement and subject satisfaction. J Drugs Dermatol. 2021;20(9):988-995. doi:10.36849/JDD.6130.

Marginal Mandibular Nerve Mapping Prior to Nonablative Radiofrequency Skin Tightening.

BACKGROUND: Aging skin and increased skin laxity is a prevalent concern of patients. Nonsurgical treatments, such as radiofrequency, are increasing in popularity due to decreased pain, downtime, and scarring. ThermiRF (Thermi, Irving, TX) is a subdermal radiofrequency treatment for tightening skin. When applying radiofrequency treatments to the neck, it is important to avoid ablating the marginal mandibular nerve and causing nerve trauma. OBJECTIVES: The purpose of this study was to locate and record the position of the marginal mandibular nerve in 72 patients undergoing subdermal radiofrequency skin tightening, to determine how often the nerve correlates to its textbook anatomic position. METHODS: Marginal mandibular nerves were located with a nerve stimulator and marked with the subject in both upright and recumbent positions. Photographs were taken and the nerve position in relation to the mandible was recorded. RESULTS: The marginal mandibular nerve was in its correct anatomic position above the mandible in 18% of patients. Nerve position did not shift between the upright and recumbent positions. Only 10% of patients had left-right nerve symmetry. CONCLUSIONS: To avoid nerve injuries, nerve mapping prior to nonablative radiofrequency treatment is recommended. The marginal mandibular nerve is not always in its correct anatomic position or symmetric to the opposing side. Its location cannot be assumed from the textbook anatomic position or from a single-side mapping.

Successful Treatment of Scalp Micropigmentation With 1064 nm Picosecond Nd:YAG Laser.

BACKGROUND AND OBJECTIVES: Scalp micropigmentation (SMP) is a useful technique that applies permanent camouflage for hair loss and scarring. However, suboptimal outcomes necessitate removal. To date, there have been no reports of SMP removal with a picosecond laser. In this report, we demonstrate the safety and efficacy of 1064 nm picosecond neodymium yttrium aluminum garnet (Nd:YAG) laser for the removal of SMP. STUDY DESIGN/MATERIALS AND METHODS: This was a case series of four patients who presented with undesirable SMP. Following induction of adequate cutaneous anesthesia, a 1064 nm picosecond Nd:YAG laser was applied to all surface areas with SMP. Post-operative care consisted of 1% dimethicone ointment applied twice daily to the affected areas for 3-7 days. Patients were evaluated after 3-4 weeks and additional treatment sessions were undertaken if necessary. RESULTS: Clearance of SMP was achieved within 1-3 treatment sessions in all cases. There were no unexpected adverse events. CONCLUSION: Picosecond Nd:YAG laser is an excellent treatment option for undesirable SMP. It is safe and effective and typically requires fewer treatment sessions than traditional tattoo removal. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

The Efficacy of Pulsed Dye Laser Pretreated With or Without Local Anesthetic on Patients Presenting With Erythema of Face, Neck, Chest, and Extremities.

BACKGROUND AND OBJECTIVES: Erythema is one of the most common cosmetic concerns and usually responds well to pulsed dye laser (PDL) treatment. As this laser can cause significant discomfort, topical anesthesia is sometimes offered. However, it is still uncertain whether topical anesthetics can affect the outcome of the laser therapy. We performed a retrospective single site study to compare the efficacy of PDL for the treatment of erythema in patients with and without pretreatment with topical anesthetic. STUDY DESIGN/MATERIALS AND METHODS: A chart review was performed and patients who presented with erythema of face, neck, chest, and extremities pretreated with topical anesthesia (23% lidocaine/7% tetracaine ointment or 7% lidocaine/7% tetracaine ointment) undergoing PDL were reviewed and compared with another group without anesthesia. Two blinded dermatologists evaluated the postlaser procedure photographs and gave an assessment compared with baseline. RESULTS: A total of 69 patient charts were reviewed. The erythema resulted from various skin conditions including telangiectasia, cherry angioma, striae, and rosacea. The mean improvement was 2.2581 in the anesthesia group and 2.2632 in the nonanesthesia group. There was no significant difference between both groups as confirmed by a noninferiority test. CONCLUSIONS: Topical anesthesia with lidocaine and tetracaine ointment do not interfere with the efficacy of the PDL. Since pain management is essential for any cosmetic procedure, the application of a local anesthetic will enhance patient comfort and satisfaction during treatment with PDL. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

A Randomized, Split-Face, Evaluator-Blind Clinical Trial Comparing Monopolar Radiofrequency Versus Microfocused Ultrasound With Visualization for Lifting and Tightening of the Face and Upper Neck.

BACKGROUND: Over the past decade, 2 major modalities for noninvasive skin tightening have emerged: monopolar capacitive-coupled radiofrequency (MRF) and microfocused ultrasound with visualization (MFU-V). Up to date, no comparative clinical trials have been performed. OBJECTIVE: To compare the efficacy and safety of MRF versus MFU-V for the lifting and tightening of the face and neck. MATERIALS AND METHODS: Twenty subjects with mild to moderate skin laxity received MFU-V over one-side of the face and MRF over the other side of the face at the same time. Subjects were followed for 6 months. RESULTS: Both MRF and MFU-V led to a decrease in the Fasil Face and Neck Laxity Grading Scale (FLR). These differences became significant at Day 30 and remained significantly improved through Days 90 and 180 in both groups. There was no statistically significant difference in the FLR Scale between MRF-treated and MFU-V-treated sides. Subjects' Global Aesthetic Improvement Scale showed improvement at Day 30, 90, and 180. CONCLUSION: Both MRF and MFU-V led to significant improvement in face and neck laxity. There were no statistical differences between MRF and MFU-V in standardized investigator measures of face and neck laxity, patient satisfaction, and adverse events.

Red Deer Umbilical Cord Lining Mesenchymal Stem Cell Extract Cream for Rejuvenation of the Face

Background: Aging is a multifactorial process that involves all components of the skin. Both intrinsic and extrinsic forces play a role in this aging process. A new patented protein mix derived from red deer umbilical cord lining stem cell conditioned media (Calecim® Multi Action Cream, CellResearch Corporation, Singapore) has been developed to improve the signs of aging. The extract is the conditioned media from umbilical cord lining mesenchymal stem cell culture in basal media and consists of a mixture, in specific proportions, of cytokines, growth factors, extracellular matrix proteins, amino acids, peptides, and other proteins. It has been developed to increase epidermal cell turnover and stimulate fibroblast function, reducing the appearance of pigmentation, fine lines, and redness, and to restore skin elasticity. Objective: The objective of this IRB-approved, prospective, randomized, double-blind, split-face, placebo-controlled clinical trial was to compare the efficacy of red deer mesenchymal stem cell extract (RCE) versus vehicle for facial rejuvenation. Methods: The trial involved 40 healthy subjects with moderate to severe facial wrinkling secondary to photodamage. One half of the face was randomized to receive topical RCE cream and vehicle cream to the other half of the face. Treatment was continued for 3 months, and evaluations were performed in a double-blind fashion. Results: Both sides of the face achieved significant improvement. Blinded investigator assessments did not detect any statistically significant differences between the two halves of the face in terms of efficacy, safety, or tolerability. Subject evaluations demonstrated superiority of the active treatment side. Conclusion: Red deer umbilical cord lining mesenchymal stem cell extract was effective in rejuvenating the aging face as demonstrated by investigator and subject measures. J Drugs Dermatol. 2019;18(4):363-366.

Clinical Correlations of Transcriptional Profile in Patients Infected With Avian Influenza H7N9 Virus.

Background: Avian influenza A (H7N9) viruses emerged in China in 2013 and caused zoonotic disease associated with a case-fatality ratio of over 30%. Transcriptional profiles in peripheral blood reflect host responses and can help to elucidate disease pathogenesis. Methods: We correlated serial blood transcriptomic profiles of patients with avian influenza A (H7N9) virus infection and determined the biological significances from the analysis. Results: We found that specific gene expression profiles in the blood were strongly correlated with the Pao 2/Fio 2 ratio and viral load in the lower respiratory tract. Cell cycle and leukocyte-related immunity were activated at the acute stage of the infection while T-cell functions and various metabolic processes were associated with the recovery phase of the illness. A transition from systemic innate to adaptive immunity was found. Conclusions: We developed a novel approach for transcriptomic analysis to identify key host responses that were strongly correlated with specific clinical and virologic parameters in patients with H7N9 infection.

Limitations of alignment-free tools in total RNA-seq quantification.

BACKGROUND: Alignment-free RNA quantification tools have significantly increased the speed of RNA-seq analysis. However, it is unclear whether these state-of-the-art RNA-seq analysis pipelines can quantify small RNAs as accurately as they do with long RNAs in the context of total RNA quantification. RESULT: We comprehensively tested and compared four RNA-seq pipelines for accuracy of gene quantification and fold-change estimation. We used a novel total RNA benchmarking dataset in which small non-coding RNAs are highly represented along with other long RNAs. The four RNA-seq pipelines consisted of two commonly-used alignment-free pipelines and two variants of alignment-based pipelines. We found that all pipelines showed high accuracy for quantifying the expression of long and highly-abundant genes. However, alignment-free pipelines showed systematically poorer performance in quantifying lowly-abundant and small RNAs. CONCLUSION: We have shown that alignment-free and traditional alignment-based quantification methods perform similarly for common gene targets, such as protein-coding genes. However, we have identified a potential pitfall in analyzing and quantifying lowly-expressed genes and small RNAs with alignment-free pipelines, especially when these small RNAs contain biological variations.

RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase.

Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel template-switching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5' to 3' gene coverage and identify more splice junctions, particularly near the 5' ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq.