Clinical Evaluation of Fiducial Marker Pre-Planning for Virtual Bronchoscopic Navigation Implantation in Lung Tumour Patients Treated With CyberKnife.

Purpose: For the treatment of invisible lung tumours with CyberKnife (CK), fiducial markers (FMs) were implanted as an internal surrogate under virtual bronchoscopic navigation (VBN). This research aims to study the benefits of introducing an additional procedure in assigning the optimal FM positions using a pre-procedure planning system and performing virtual simulation before implantation. The objectives were 1) to reduce the duration of the FM implantation procedure, 2) to reduce the radiation exposure in dose area product (DAP) (dGy*cm2) to patients, and 3) to increase the number of FMs implanted around the tumour. Methods and Materials: This study is retrospective, single-centre, and observational in nature. A total of 32 patients were divided into two groups. In Group 1, 18 patients underwent conventional VBN FM implantation. In Group 2, 14 patients underwent additional pre-procedure planning and simulation. The steps of pre-procedure planning include 1) importing CT images into the treatment planning system (Eclipse, Varian Medical Systems, Inc.) and delineating five to six FMs in their ideal virtual positions and 2) copying the FM configuration into VBN planning software (LungPoint Bronchus Medical, Inc.) for verification and simulation. Finally, the verified FMs were deployed through VBN with the guidance of the LungPoint planning software. Results: A total of 162 FMs were implanted among 35 lesions in 32 patients aged from 37 to 92 (median = 66; 16 men and 16 women). Results showed that 1) the average FM insertion time was shortened from 41 min (SD = 2.05) to 23 min (SD = 1.25), p = 0.00; 2) the average absorbed dose of patients in DAP was decreased from 67.4 cGy*cm2 (SD = 14.48) to 25.3 cGy*cm2 (SD = 3.82), p = 0.01 (1-tailed); and 3) the average number of FMs implanted around the tumour was increased from 4.7 (SD = 0.84) to 5.6 (SD = 0.76), p = 0.00 (1-tailed). Conclusion: Pre-procedure planning reduces the FM implantation duration from 41.1 to 22.9 min, reduces the radiation exposure in DAP from 67.4 to 25.3 dGy*cm2, and increases the number of FMs inserted around the tumour from 4.7 to 5.6.

Respiratory 4D-Gating F-18 FDG PET/CT Scan for Liver Malignancies: Feasibility in Liver Cancer Patient and Tumor Quantitative Analysis.

PURPOSE: To evaluate the potential clinical role and effectiveness of respiratory 4D-gating F-18 FDG PET/CT scan for liver malignancies, relative to routine (3D) F-18 FDG PET/CT scan. MATERIALS AND METHODS: This study presented a prospective clinical study of 16 patients who received F-18 FDG PET/CT scan for known or suspected malignant liver lesions. Ethics approvals were obtained from the ethics committees of the Hong Kong Baptist Hospital and The Hong Kong Polytechnic University. Liver lesions were compared between the gated and ungated image sets, in terms of 1) volume measurement of PET image, 2) accuracy of maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), and 3) accuracy of total lesion glycoses (TLG). Statistical analysis was performed by using a two-tailed paired Student t-test and Pearson correlation test. RESULTS: The study population consisted of 16 patients (9 males and 7 females; mean age of 65) with a total number of 89 lesions. The SUVmax and SUVmean measurement of the gated PET images was more accurate than that of the ungated PET images, compared to the static reference images. An average of 21.48% (p < 0.001) reduction of the tumor volume was also observed. The SUVmax and SUVmean of the gated PET images were improved by 19.81% (p < 0.001) and 25.53% (p < 0.001), compared to the ungated PET images. CONCLUSIONS: We have demonstrated the feasibility of implementing 4D PET/CT scan for liver malignancies in a prospective clinical study. The 4D PET/CT scan for liver malignancies could improve the quality of PET image by improving the SUV accuracy of the lesions and reducing image blurring. The improved accuracy in the classification and identification of liver tumors with 4D PET image would potentially lead to its increased utilization in target delineation of GTV, ITV, and PTV for liver radiotherapy treatment planning in the future.

Comparison of dosimetric impact of intra-fractional setup discrepancy between multiple- and single-isocenter approaches in linac-based stereotactic radiotherapy of multiple brain metastases.

INTRODUCTION: Treatment of multiple brain metastases by linac-based stereotactic radiotherapy (SRT) can employ either a multiple-isocenter (MI) or single-isocenter (SI) approach. The purposes of this study were to evaluate the dosimetric results of MI and SI approaches and compare the impacts of intra-fractional setup discrepancies on the robustness of respective approaches using isocenter shifts, whether the same magnitude of translational and rotational effects could lead to a significant difference between the two approaches. METHODS: Twenty-two patients with multiple brain metastases treated by linac-based SRT were recruited. Treatment plans were computed with both the MI and SI approaches. For the MI approach, the isocenter was located at the geometric center of each planning target volumes (PTVs), whereas the isocenter of the SI approach was located midway between the PTV centroids. To simulate the intra-fractional errors, isocenter displacements including translational and rotational shifts were hypothetically applied. Apart from the dosimetric outcomes of the two approaches, the impact of the isocenter shifts on PTVs and organs at risk (OARs) were recorded in terms of the differences (δ) in dose parameters relative to the reference plan and was then compared between the MI and SI approaches. RESULTS: Both MI and SI plans met the plan acceptance criteria. The mean Paddick conformity index (Paddick CI) and Dmax of most OARs between MI and SI plans did not show a significant difference, except that higher doses to the left optic nerve and optic chiasm were found in SI plans (p = 0.03). After the application of the isocenter shifts, δCI increased with an increase in the magnitude of the isocenter shift. When comparing between MI and SI plans, the δCIs were similar (p > 0.05) for all extents of translational shifts, but δCIs were significantly higher in SI plans after application of all rotations particularly ±1.5° and ±2.0° shifts. Despite the result that the majority of δDMax of OARs were higher in the SI plans, only the differences in the left optic nerve and chiasm showed generally consistent significance after both translational ≥±1 mm and rotational shifts of ≥±1 ∘ $^\circ $ . CONCLUSION: Both MI and SI approaches could produce clinically acceptable plans. However, isocenter shifts brought dosimetric impacts to both MI and SI approaches and the effects increased with the increase of the shift magnitude. Although similar impacts were shown in plans of both approaches after translational isocenter shift, SI plans were relatively more vulnerable than MI plans to rotational shifts.

Aspects of the Neurospora crassa Sulfur Starvation Response Are Revealed by Transcriptional Profiling and DNA Affinity Purification Sequencing.

Accurate nutrient sensing is important for rapid fungal growth and exploitation of available resources. Sulfur is an important nutrient source found in a number of biological macromolecules, including proteins and lipids. The model filamentous fungus Neurospora crassa is capable of utilizing sulfur found in a variety of sources from amino acids to sulfate. During sulfur starvation, the transcription factor CYS-3 is responsible for upregulation of genes involved in sulfur uptake and assimilation. Using a combination of RNA sequencing and DNA affinity purification sequencing, we performed a global survey of the N. crassa sulfur starvation response and the role of CYS-3 in regulating sulfur-responsive genes. The CYS-3 transcription factor bound the promoters and regulated genes involved in sulfur metabolism. Additionally, CYS-3 directly activated the expression of a number of uncharacterized transporter genes, suggesting that regulation of sulfur import is an important aspect of regulation by CYS-3. CYS-3 also directly regulated the expression of genes involved in mitochondrial electron transfer. During sulfur starvation, genes involved in nitrogen metabolism, such as amino acid and nucleic acid metabolic pathways, along with genes encoding proteases and nucleases that are necessary for scavenging nitrogen, were activated. Sulfur starvation also caused changes in the expression of genes involved in carbohydrate metabolism, such as those encoding glycosyl hydrolases. Thus, our data suggest a connection between sulfur metabolism and other aspects of cellular metabolism. IMPORTANCE Identification of nutrients present in the environment is a challenge common to all organisms. Sulfur is an important nutrient source found in proteins, lipids, and electron carriers that are required for the survival of filamentous fungi such as Neurospora crassa. Here, we transcriptionally profiled the response of N. crassa to characterize the global response to sulfur starvation. We also used DNA affinity purification sequencing to identify the direct downstream targets of the transcription factor responsible for regulating genes involved in sulfur uptake and assimilation. Along with genes involved in sulfur metabolism, this transcription factor regulated a number of uncharacterized transporter genes and genes involved in mitochondrial electron transfer. Our data also suggest a connection between sulfur, nitrogen, and carbon metabolism, indicating that the regulation of a number of metabolic pathways is intertwined.

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus.

Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.

A longitudinal study on parotid and submandibular gland changes assessed by magnetic resonance imaging and ultrasonography in post-radiotherapy nasopharyngeal cancer patients.

OBJECTIVES: With regard to the intensity modulated radiotherapy (IMRT) of nasopharyngeal carcinoma (NPC) patients, this longitudinal study evaluated the radiation-induced changes in the parotid and submandibular glands in terms of gland size, echogenicity and haemodynamic parameters. METHODS: 21 NPC patients treated by IMRT underwent MRI and ultrasound scans before radiotherapy, and at 6, 12, 18 and 24 months after treatment. Parotid and submandibular gland volumes were measured from the MRI images, whereas the parotid echogenicity and haemodynamic parameters including the resistive index, pulsatility index, peak systolic velocity and end diastolic velocity were evaluated by ultrasonography. Trend lines were plotted to show the pattern of changes. The correlations of gland doses and the post-RT changes were also studied. RESULTS: The volume of the parotid and submandibular glands demonstrated a significant drop from pre-RT to 6 months post-RT. The parotid gland changed from hyperechoic before RT to either isoechoic or hypoechoic after treatment. The resistive index and pulsatility index decreased from pre-RT to 6 month post-RT, then started to increase at 12 month time interval. Both peak systolic velocity and end diastolic velocity increased after 6 months post-RT then followed a decreasing trend up to 24 months post-RT. There was mild correlation between post-RT gland dose and gland volume, but not with haemodynamic changes. CONCLUSIONS: Radiation from IMRT caused shrinkage of parotid and submandibular glands in NPC patients. It also changed the echogenicity and vascular condition of the parotid gland. The most significant changes were observed at 6 months after radiotherapy. ADVANCES IN KNOWLEDGE: It is the first paper that reports on the longitudinal changes of salivary gland volume, echogenicity and haemodynamic parameters altogether in NPC patients after radiotherapy. The results are useful for the prediction of glandular changes that is associated with xerostomia, which help to provide timely management of the complication when the patients attend follow-up visits.

Radiation induced temporal lobe necrosis in nasopharyngeal cancer patients after radical external beam radiotherapy.

Radiation-induced temporal lobe necrosis (TLN) is one of the late post-radiotherapy complications in nasopharyngeal cancer (NPC) patients. Since NPC is common to have skull base infiltration, irradiation of the temporal lobes is inevitable despite the use of the more advanced intensity-modulated radiotherapy (IMRT). Moreover, the diagnosis and treatment of TLN remain challenging. In this review, we discuss the diagnosis of TLN with conventional and advanced imaging modalities, onset and predictive parameters of TLN development, the impact of IMRT on TLN in terms of incidence and dosimetric analyzes, and the recent advancements in the treatment of TLN.

Hypoxia-Induced Epithelial-Mesenchymal Transition in Cancers: HIF-1α and Beyond.

Metastasis is the main cause of cancer-related mortality. Although the actual process of metastasis remains largely elusive, epithelial-mesenchymal transition (EMT) has been considered as a major event in metastasis. Besides, hypoxia is common in solid cancers and has been considered as an important factor for adverse treatment outcomes including metastasis. Since EMT and hypoxia potentially share several signaling pathways, many recent studies focused on investigate the issue of hypoxia-induced EMT. Among all potential mediators of hypoxia-induced EMT, hypoxia-inducible factor-1α (HIF-1α) has been studied extensively. Moreover, there are other potential mediators that may also contribute to the process. This review aims to summarize the recent reports on hypoxia-induced EMT by HIF-1α or other potential mediators and provide insights for further investigations on this issue. Ultimately, better understanding of hypoxia-induced EMT may allow us to develop anti-metastatic strategies and improve treatment outcomes.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

JNK Pathway Mediates Low Oxygen Level Induced Epithelial-Mesenchymal Transition and Stemness Maintenance in Colorectal Cancer Cells.

(1) Background: Epithelial-mesenchymal transition (EMT) and cancer cell stemness maintenance (SM) are important factors for cancer metastasis. Although hypoxia has been considered as a possible factor for EMT induction and promotion of SM, studies in this area, apart from hypoxia-inducible factor (HIF) pathways and severe hypoxia, are scant. This study aimed to evaluate the effects of different oxygen levels on EMT induction and SM and elucidate the signaling pathways involved in colorectal cancer cells. (2) Methods: Cell morphological analysis, migration assay, immunofluorescence staining of cytoskeleton and Western blotting were performed on human colorectal cancer cells HT-29, DLD-1, and SW-480 cultured at 1%, 10%, and normal (21%) O2 levels. The role played by c-Jun N-terminal kinase (JNK) was evaluated through the use of the specific JNK inhibitor SP600125. (3) Results: This study evaluated 1% and 10% O2 are possible conditions for EMT induction and SM. This study also demonstrated the partial relieve of EMT induction and SM by SP600125, showing the importance of the JNK pathway in these processes. Furthermore, this study proposed a novel pathway on the regulation of Akt by JNK-c-Jun. (4) Conclusions: This study suggests 10% O2 as another possible condition for EMT induction, and SM and JNK pathways play important roles in these processes through multiple factors. Inhibition of JNK could be explored as treatment for inhibiting metastasis in colorectal cancer cells.

A Review on the Assessment of Radiation Induced Salivary Gland Damage After Radiotherapy.

Head and neck cancers are common in Southern China including Hong Kong. Intensity modulated radiotherapy has been the treatment of choice for these patients. Although radiotherapy provides good local control, radiotherapy treatment side-effects are still inevitable due to close proximity of the organs at risk from the target volume. Xerostomia, which is caused due to the damage of salivary glands, is one of the main radiation induced toxicities in post-radiotherapy head and neck patients. This review article discusses the methods for the assessing of radiation induced salivary gland changes including the gland morphology and saliva flow rate. The discussion also includes the recovery of the salivary gland after radiotherapy and how it is affected by the dose. It is expected that the future direction in monitoring the recovery of salivary glands will focus in cellular or molecular levels, and the development of imaging biomarker.

A dosimetric comparison of the use of equally spaced beam (ESB), beam angle optimization (BAO), and volumetric modulated arc therapy (VMAT) in head and neck cancers treated by intensity modulated radiotherapy.

INTRODUCTION: Previous studies have shown that the beam arrangement had significant influence on plan quality in intensity modulated radiotherapy (IMRT). This study aimed to evaluate the dosimetric performance of beam arrangement methods by employing equally spaced beams (ESB), beam angle optimization (BAO), and volumetric modulated arc therapy (VMAT) in the planning of five types of head and neck (H&N) cancers treated by IMRT. METHODS: Five plans of different beam arrangement methods were optimized for 119 H&N cancer patients with the prescription of 66-70 Gy for high-risk planning target volume (PTV), 60 Gy for intermediate risk PTV, 54 Gy for low-risk PTV using a simultaneously integrated boost method. The five-beam arrangement methods were: ESB, coplanar BAO (BAOc), noncoplanar BAO (BAOnc), two-arc VMAT (VMAT2), and three-arc VMAT (VMAT3). The H&N cancers included cancers of nasopharynx, oral cavity, larynx, maxillary sinus, and parotid. Although the partial arc VMAT could be used in cases where the PTVs were situated at one side of the head such as the parotid, this arrangement was not included because it was intended to include only the beam arrangements that were applicable to all the types of head and neck cancers in the study. The plans were evaluated using a "figure-of-merit" known as uncomplicated target conformity index (UTCI). In addition, PTV conformation number and homogeneity index, normal tissue integral dose, and organ at risk (OAR) doses were also used. The mean values of these parameters were compared among the five plans. RESULTS: All treatment plans met the preset dose requirements for the target volumes and OARs. For nasopharyngeal cancer, VMAT3 and BAOnc demonstrated significantly higher UTCI. For cancer of oral cavity, most beam arrangement showed similar UTCI except ESB, which was relatively lower. For cancer of larynx, there was no significant difference in UTCI among the five-beam arrangement methods. For cancers of maxillary sinus and parotid gland, the two BAO methods showed marginally higher UTCI among all the five methods. CONCLUSION: Individual methods showed dosimetric advantages on certain aspects, and the UTCI of the BAO treatment plans are marginally greater in the case of maxillary sinus and parotid gland. However, if treatment time was included into consideration, VMAT plans would be recommended for cancers of the nasopharynx, oral cavity, and larynx.

Intrafractional motion management in external beam radiotherapy.

The recent advancements in radiotherapy technologies have made delivery of the highly conformal dose to the target volume possible. With the increasing popularity of delivering high dose per fraction in modern radiotherapy schemes such as in stereotactic body radiotherapy and stereotactic body ablative therapy, high degree of treatment precision is essential. In order to achieve this, we have to overcome the potential difficulties caused by patient instability due to immobilization problems; patient anxiety and random motion due to prolonged treatment time; tumor deformation and baseline shift during a treatment course. This is even challenging for patients receiving radiotherapy in the chest and abdominal regions because it is affected by the patient's respiration which inevitably leads to tumor motion. Therefore, monitoring of intrafractional motion has become increasingly important in modern radiotherapy. Major intrafractional motion management strategies including integration of respiratory motion in treatment planning; breath-hold technique; forced shallow breathing with abdominal compression; respiratory gating and dynamic real-time tumor tracking have been developed. Successful intrafractional motion management is able to reduce the planning target margin and ensures planned dose delivery to the target and organs at risk. Meanwhile, the emergency of MRI-linear accelerator has facilitated radiation-free real-time monitoring of soft tissue during treatment and could be the future modality in motion management. This review article summarizes the various approaches that deal with intrafractional target, organs or patient motion with discussion of their advantages and limitations. In addition, the potential future advancements including MRI-based tumor tracking are also discussed.

Dynamics of oxygen level-driven regulators in modulating autophagy in colorectal cancer cells.

Colorectal cancer is a common cancer with metachronous distant metastases still threatening overall survival. Tumor oxygen level influences tumor radiosensitivity in relation to autophagy and apoptosis. The objective of this study is to evaluate the expression and interaction between multiple key regulators in different oxygen levels. Human colorectal adenocarcinoma HT-29 cells were cultured in 1% or 10% oxygen level and irradiated by 2 Gy with different incubation time. Autophagy key regulators, AMPK, HIFs and JNK were evaluated by Western blot. Sequential autophagy key regulator activation was observed in the order of AMPK, HIF-1α, HIF-2α and JNK. 10% oxygen level could promote autophagy with similar degree of autophagy activation as 1% oxygen level in 48-h while irradiation could slightly inhibit autophagy. The results of this study supported prior evaluation of oxygen level and autophagy regulators for improving treatment efficacy and indicated the possible directions in developing individualized radiotherapy by selective targeting of hypoxic regions.

A Review on the Special Radiotherapy Techniques of Colorectal Cancer.

Colorectal cancer is one of the commonest cancers worldwide. Radiotherapy has been established as an indispensable component of treatment. Although conventional radiotherapy provides good local control, radiotherapy treatment side-effects, local recurrence and distant metastasis remain to be the concerns. With the recent technological advancements, various special radiotherapy treatment options have been offered. This review article discusses the recently-developed special radiotherapy treatment modalities for various conditions of colorectal cancer ranging from early stage, locally advanced stage, recurrent, and metastatic diseases. The discussion focuses on the areas of feasibility, local control, and survival benefits of the treatment modalities. This review also provides accounts of the future direction in radiotherapy of colorectal cancer with emphasis on the coming era of personalized radiotherapy.

Development of a normal tissue complication probability (NTCP) model for radiation-induced hypothyroidism in nasopharyngeal carcinoma patients.

BACKGROUND: The objectives of this study were to build a normal tissue complication probability (NTCP) model of radiation-induced hypothyroidism (RHT) for nasopharyngeal carcinoma (NPC) patients and to compare it with other four published NTCP models to evaluate its efficacy. METHODS: Medical notes of 174 NPC patients after radiotherapy were reviewed. Biochemical hypothyroidism was defined as an elevated level of serum thyroid-stimulating hormone (TSH) value with a normal or decreased level of serum free thyroxine (fT4) after radiotherapy. Logistic regression with leave-one-out cross-validation was performed to establish the NTCP model. Model performance was evaluated and compared by the area under the receiver operating characteristic curve (AUC) in our NPC cohort. RESULTS: With a median follow-up of 24 months, 39 (22.4%) patients developed biochemical hypothyroidism. Gender, chemotherapy, the percentage thyroid volume receiving more than 50 Gy (V50), and the maximum dose of the pituitary (Pmax) were identified as the most predictive factors for RHT. A NTCP model based on these four parameters were developed. The model comparison was made in our NPC cohort and our NTCP model performed better in RHT prediction than the other four models. CONCLUSIONS: This study developed a four-variable NTCP model for biochemical hypothyroidism in NPC patients post-radiotherapy. Our NTCP model for RHT presents a high prediction capability. TRIAL REGISTRATION: This is a retrospective study without registration.

Regulation of Cell-to-Cell Communication and Cell Wall Integrity by a Network of MAP Kinase Pathways and Transcription Factors in Neurospora crassa.

Maintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such as Neurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP kinase (MAPK) pathways are essential for communication and cell fusion in N. crassa: the cell wall integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross-talk between the MAK-1 and MAK-2 pathways, which is likely necessary for coordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAPK pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, but it is unclear what targets ADV-1. We did RNA sequencing on Δadv-1, Δpp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting the in vitro method of DNA-affinity purification sequencing for N. crassa To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression of adv-1 was sufficient to fully suppress the phenotype of the Δpp-1 mutant and partially suppress the phenotype of the Δmak-1 mutant. Collectively, our data demonstrate that the MAK-1/ADV-1 and MAK-2/PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa.

BACKGROUND: Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction. RESULTS: The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of l-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif. CONCLUSIONS: PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.

A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism.

In Neurospora crassa, the transcription factor COL-26 functions as a regulator of glucose signaling and metabolism. Its loss leads to resistance to carbon catabolite repression. Here, we report that COL-26 is necessary for the expression of amylolytic genes in N. crassa and is required for the utilization of maltose and starch. Additionally, the Δcol-26 mutant shows growth defects on preferred carbon sources, such as glucose, an effect that was alleviated if glutamine replaced ammonium as the primary nitrogen source. This rescue did not occur when maltose was used as a sole carbon source. Transcriptome and metabolic analyses of the Δcol-26 mutant relative to its wild type parental strain revealed that amino acid and nitrogen metabolism, the TCA cycle and GABA shunt were adversely affected. Phylogenetic analysis showed a single col-26 homolog in Sordariales, Ophilostomatales, and the Magnaporthales, but an expanded number of col-26 homologs in other filamentous fungal species. Deletion of the closest homolog of col-26 in Trichoderma reesei, bglR, resulted in a mutant with similar preferred carbon source growth deficiency, and which was alleviated if glutamine was the sole nitrogen source, suggesting conservation of COL-26 and BglR function. Our finding provides novel insight into the role of COL-26 for utilization of starch and in integrating carbon and nitrogen metabolism for balanced metabolic activities for optimal carbon and nitrogen distribution.

Deciphering the Regulatory Network between the SREBP Pathway and Protein Secretion in Neurospora crassa.

Sterol regulatory element binding proteins (SREBPs) are conserved from yeast to mammalian cells and function in the regulation of sterol homeostasis. In fungi, the SREBP pathway has been implicated in the adaptation to hypoxia and in virulence. In Neurospora crassa and Trichoderma reesei, the SREBP pathway also negatively regulates protein secretion under lignocellulolytic conditions. Here we utilized global transcriptional profiling combined with genetic and physiological analyses to address the regulatory link between the SREBP pathway and protein secretion in N. crassa Our results demonstrated that the function of the SREBP pathway in ergosterol biosynthesis and adaptation to hypoxia was conserved in N. crassa Under lignocellulolytic conditions, the SREBP pathway was highly activated, resulting in the reduced expression of lytic polysaccharide monooxygenases, which require molecular oxygen for catalytic activity. Additionally, activation of the SREBP pathway under lignocellulolytic conditions repressed a set of genes predicted to be involved in the endoplasmic reticulum stress response. Here we show that the inability of a hac-1 mutant, which bears a deletion of the major regulator of the unfolded protein response (UPR), to efficiently produce cellulases and utilize cellulose was suppressed by mutations in the SREBP pathway. The analyses presented here demonstrated new SREBP pathway functions, including linkages to the UPR, and provide new clues for genetic engineering of filamentous fungi to improve their production of extracellular proteins.IMPORTANCE The role of SREBP transcription factors in the regulation of sterol biosynthesis is conserved from humans to yeast. In filamentous fungi, this pathway regulates the secretion of lignocellulolytic enzymes during plant biomass deconstruction. Here we show that the SREBP pathway in Neurospora crassa regulates the production of specific cellulases, lytic polysaccharide monooxygenases that utilize molecular oxygen. Via global transcriptional profile and genetic analyses, a relationship between the SREBP pathway and the unfolded protein response (UPR) pathway was revealed, suggesting a regulatory interplay of these two pathways in the trafficking of plant biomass-degrading enzymes. These findings have implications for our understanding of the cross talk of the SREBP and UPR pathways in other organisms and will guide the rational engineering of fungal strains to improve cellulolytic enzyme production.